Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion

The process of erythrocyte invasion by merozoites of Plasmodium falciparum involves multiple steps, including the formation of a moving junction between parasite and host cell, and it is characterised by the redundancy of many of the receptor-ligand interactions involved. Several parasite proteins that interact with erythrocyte receptors or participate in other steps of invasion are encoded by small subtelomerically located gene families of four to seven members. We report here that members of the eba, rhoph1/clag, acbp, and pfRh multigene families exist in either an active or a silenced state. In the case of two members of the rhoph1/clag family, clag3.1 and clag3.2, expression was mutually exclusive. Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic. This was demonstrated for eba-140. Our data demonstrate that variant or mutually exclusive expression and epigenetic silencing in Plasmodium are not unique to genes such as var, which encode proteins that are exported to the surface of the erythrocyte, but also occur for genes involved in host cell invasion. Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.     

The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum

Plasmodium falciparum is the most virulent causative agent of malaria in man accounting for 80% of all malarial infections and 90% of the one million annual deaths attributed to malaria. P. falciparum is a unicellular, Apicomplexan parasite, that spends part of its lifecycle in the mosquito and part in man and it has evolved a special form of motility that enables it to burrow into animal cells, a process termed “host cell invasion”. The acute, life threatening, phase of malarial infection arises when the merozoite form of the parasite undergoes multiple cycles of red blood cell invasion and rapid proliferation. Here, we discuss the molecular machinery that enables malarial parasites to invade red blood cells and we focus particularly on the ATP-driven acto-myosin motor that powers invasion.     

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.     

Plasmodium falciparum: structural and functional domains of the mature-parasite-infected erythrocyte surface antigen

The mature parasite-infected erythrocyte surface antigen (MESA) is a protein exported to the membrane skeleton of the infected red cell, where it forms a strong noncovalent interaction with the host red cell protein, protein 4.1. The complete gene structure of MESA from the Ugandan isolate Palo Alto is described. Comparison to the previously reported MESA sequence from the Papua New Guinean cloned line D10 reveals strong conservation of the general gene structure of a short first exon and a long second exon. The exact exon/intron boundaries were determined by the generation and sequencing of a cDNA from this region. The MESA gene from both isolates consists of seven blocks of repeats that are identical in order. Repeat blocks are conserved to a high degree; however, differences are noted in most blocks in the form of scattered mutations or differences in repeat numbers. Previous work had shown that synthetic peptides spanning a 19-residue region could inhibit the binding of MESA to protein 4.1. Removal of this region from MESA almost completely abolished the binding of MESA to IOVs. Sequencing of this region from a number of laboratory and field isolates demonstrates complete conservation of the cytoskeletal binding domain and flanking sequences.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C–EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine‐rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)–EPCR interaction. Overall, there was a positive correlation between CIDRα1–EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti‐EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.     

A model for the progression of receptor-ligand interactions during erythrocyte invasion by Plasmodium falciparum

Multiple and seemingly sequential interactions between parasite ligands and their receptors on host erythrocytes are an essential precursor to invasion by the obligate intracellular pathogen, Plasmodium falciparum. Consequently, identification and characterisation of the specific effectors that facilitate these recognition events are of special interest for the development of novel therapeutic and prophylactic solutions to malaria. There have been many recent advances regarding the identification of host-parasite receptor-ligand pairs, however the precise function and temporal aspects of these interactions are far from resolved. This review provides an update on the current details of these interactions to place them in sequence and super impose them upon the known kinetic events of invasion.     

Role of Ca2+/calmodulin-PfPKB signaling pathway in erythrocyte invasion by Plasmodium falciparum

Molecular mechanisms by which signaling pathways operate in the malaria parasite and control its development are promiscuous. Recently, we reported the identification of a signaling pathway in Plasmodium falciparum, which involves activation of protein kinase B-like enzyme (PfPKB) by calcium/calmodulin (Vaid, A., and Sharma, P. (2006) J. Biol. Chem. 281, 27126-27133). Studies carried out to elucidate the function of this pathway suggested that it may be important for erythrocyte invasion. Blocking the function of the upstream activators of this pathway, calmodulin and phospholipase C, resulted in impaired invasion. To evaluate if this signaling cascade controls invasion by regulating PfPKB, inhibitors against this kinase were developed. PfPKB inhibitors dramatically reduced the ability of the parasite to invade erythrocytes. Furthermore, we demonstrate that PfPKB associates with actin-myosin motor and phosphorylates PfGAP45 (glideosome-associated protein 45), one of the important components of the motor complex, which may help explain its role in erythrocyte invasion.     

Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane

The intraerythrocytic developmental stages of the malaria parasite Plasmodium falciparum are responsible for the clinical symptoms associated with malaria tropica. The non-infected human erythrocyte is a terminally differentiated cell that is unable to synthesize proteins and lipids de novo, and it is incapable of importing a number of solutes that are essential for parasite proliferation. Approximately 12-15 h after invasion the parasitized cell undergoes a marked increase in its permeability to a variety of different solutes present in the extracellular milieu. The increase is due to the induction in the erythrocyte membrane of 'new permeability pathways' which have been characterized in some detail in terms of their transport and electrophysiological properties, but which are yet to be defined at a molecular level. Here we show that these pathways are resistant to trypsin but are abolished by treatment of intact infected erythrocytes with chymotrypsin. On resuspension of chymotrypsinized cells in chymotrypsin-free medium the pathways progressively reappear, a process that can be inhibited by cytotoxic agents, and by brefeldin A which inhibits protein secretion. Our results provide evidence for the involvement of parasite encoded proteins in the generation of the pathways, either as components of the pathways themselves or as auxiliary factors.     

Evidence that the erythrocyte invasion ligand PfRh2 is a target of protective immunity against Plasmodium falciparum malaria

Abs targeting blood-stage Ags of Plasmodium falciparum are important in acquired immunity to malaria, but major targets remain unclear. The P. falciparum reticulocyte-binding homologs (PfRh) are key ligands used by merozoites during invasion of erythrocytes. PfRh2a and PfRh2b are functionally important members of this family and may be targets of protective immunity, but their potential role in human immunity has not been examined. We expressed eight recombinant proteins covering the entire PfRh2 common region, as well as PfRh2a- and PfRh2b-specific regions. Abs were measured among a cohort of 206 Papua New Guinean children who were followed prospectively for 6 mo for reinfection and malaria. At baseline, Abs were associated with increasing age and active infection. High levels of IgG to all PfRh2 protein constructs were strongly associated with protection from symptomatic malaria and high-density parasitemia. The predominant IgG subclasses were IgG1 and IgG3, with little IgG2 and IgG4 detected. To further understand the significance of PfRh2 as an immune target, we analyzed PfRh2 sequences and found that polymorphisms are concentrated in an N-terminal region of the protein and seem to be under diversifying selection, suggesting immune pressure. Cluster analysis arranged the sequences into two main groups, suggesting that many of the haplotypes identified may be antigenically similar. These findings provide evidence suggesting that PfRh2 is an important target of protective immunity in humans and that Abs act by controlling blood-stage parasitemia and support its potential for 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 (Ca²⁺), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca²⁺ are not completely understood. Here, we show that a P. falciparum Ca²⁺‐dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca²⁺‐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 Ca²⁺‐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.     

Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite.