Plasmodium Falciparum Merozoite Invasion Research Articles

The Cellular and Molecular Interaction Between Erythrocytes and Plasmodium falciparum Merozoites.

Plasmodium falciparum is the most lethal human malaria parasite, partly due to its genetic variability and ability to use multiple invasion routes via its binding to host cell surface receptors. The parasite extensively modifies infected red blood cell architecture to promote its survival which leads to increased cell membrane rigidity, adhesiveness and permeability. Merozoites are initially released from infected hepatocytes and efficiently enter red blood cells in a well-orchestrated process that involves specific interactions between parasite ligands and erythrocyte receptors; symptoms of the disease occur during the life-cycle’s blood stage due to capillary blockage and massive erythrocyte lysis. Several studies have focused on elucidating molecular merozoite/erythrocyte interactions and host cell modifications; however, further in-depth analysis is required for understanding the parasite’s biology and thus provide the fundamental tools for developing prophylactic or therapeutic alternatives to mitigate or eliminate Plasmodium falciparum-related malaria. This review focuses on the cellular and molecular events during Plasmodium falciparum merozoite invasion of red blood cells and the alterations that occur in an erythrocyte once it has become infected.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry.

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

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.

Plasmodium falciparum Merozoite Invasion Is Inhibited by Antibodies that Target the PfRh2a and b Binding Domains

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes.

Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4

The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections.

Antibody-Mediated Growth Inhibition of Plasmodium falciparum: Relationship to Age and Protection from Parasitemia in Kenyan Children and Adults

BackgroundAntibodies that impair Plasmodium falciparum merozoite invasion and intraerythrocytic development are one of several mechanisms that mediate naturally acquired immunity to malaria. Attempts to correlate anti-malaria antibodies with risk of infection and morbidity have yielded inconsistent results. Growth inhibition assays (GIA) offer a convenient method to quantify functional antibody activity against blood stage malaria.MethodsA treatment-time-to-infection study was conducted over 12-weeks in a malaria holoendemic area of Kenya. Plasma collected from healthy individuals (98 children and 99 adults) before artemether-lumefantrine treatment was tested by GIA in three separate laboratories.ResultsMedian GIA levels varied with P. falciparum line (D10, 8.8%; 3D7, 34.9%; FVO, 51.4% inhibition). The magnitude of growth inhibition decreased with age in all P. falciparum lines tested with the highest median levels among children <4 years compared to adults (e.g. 3D7, 45.4% vs. 30.0% respectively, p = 0.0003). Time-to-infection measured by weekly blood smears was significantly associated with level of GIA controlling for age. Upper quartile inhibition activity was associated with less risk of infection compared to individuals with lower levels (e.g. 3D7, hazard ratio = 1.535, 95% CI = 1.012–2.329; p = 0.0438). Various GIA methodologies had little effect on measured parasite growth inhibition.ConclusionPlasma antibody-mediated growth inhibition of blood stage P. falciparum decreases with age in residents of a malaria holoendemic area. Growth inhibition assay may be a useful surrogate of protection against infection when outcome is controlled for age.

Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion

This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 ( 41MYNNDKILSKNEVDTNIESN 60) and 31178 ( 101YDIQATYQFPSTSGGNNVIP 120) in the amino terminal region and 31191 ( 361EIDSTINNLVQEMIHLFSNNY 380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 μg ml −1 concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.

Two MSA 2 peptides that bind to human red blood cells are relevant to Plasmodium falciparum merozoite invasion.

Plasmodium falciparum merozoite membrane surface antigen 2 (MSA2) has been associated with the development of protective immunity against malaria. MSA2 antibodies were able to inhibit in vitro merozoite invasion. In our search for experimental evidence concerning the participation of MSA2 in merozoite invasion, 40 peptides were synthesized according to sequences reported for the CAMP and FC27 prototype Plasmodium strains. These peptides were purified, 125I-radiolabeled and tested for their ability to bind to erythrocytes. Two MSA2 synthetic peptides with high specific binding to human erythrocytes were found. The peptide coded 4044 (KNESKYSNTFINNAYNMSIR), located in the MSA2 N-terminal conserved region, has an affinity coefficient of 72 nM and showed a positive cooperativity for the receptor-ligand interaction. The other peptide, coded 4053 (NPNHKNAETNPKGKGEVQKP) and located in the central variable region of MSA2, has an affinity coefficient of 49nM and also showed a positive cooperativity for the receptor-ligand interaction. The binding capacity of these peptides is affected by erythrocytes treated with neuraminidase and trypsin, but it is not affected by chymotrypsin. Both of these sequences inhibit in vitro erythrocyte parasite invasion by up to 95% suggesting that they have an important role in the parasite's invasion process. Furthermore, as published previously [A. Saul et al. (1992) J. Immunol., 148, 208-211], a protective B epitope is included in the 4044 peptide sequence.

Plasmodium falciparum merozoite invasion inhibition by cytokine primed human neutrophils

Plasmodium falciparum merozoite invasion inhibition by cytokine primed human neutrophils

Plasmodium falciparum: Analysis of the Cytoadherence Inhibition of the Human Monoclonal Antibody 33G2 and of Antibodies Reactive with Antigen Pf332

The capacity of a human monoclonal antibody (MAb 33G2) to interfere in vitro both with Plasmodium falciparum merozoite invasion and cytoadherence of infected erythrocytes to melanoma cells has been reported. MAb 33G2 cross-reacts with several P. falciparum antigens but shows highest reactivity with repeated sequences in the asexual blood stage antigen Pf332. This study was conducted in order to further analyze the cytoadherence inhibition mediated by MAb 33G2 and to evaluate the relative contribution of antibodies to Pf332 in the inhibitory activity of immunoglobulins from P. falciparum immune donors. We show here that MAb 33G2 inhibits cytoadherence of infected erythrocytes (PRBCs) with similar efficiency independently of the strain of parasite, while the inhibitory capacity of immunoglobulin fractions from Liberian immune donors was restricted to some strains only. There appears to be no correlation between the reactivity with Pf332 of immunoglobulin preparations from different donors and their capacity to inhibit cytoadherence of PRBCs to melanoma cells. In contrast to MAb 33G2, polyclonal antibodies affinity purified on the Pf332 peptide containing the epitope seen by the MAb showed little or no inhibition of cytoadherence of infected erythrocytes.

Effect of different fractions of heparin on Plasmodium falciparum merozoite invasion of red blood cells in vitro.

Heparin and various heparin fractions were separated according to differences in molecular weight or affinity for antithrombin III and used for the inhibition of Plasmodium falciparum merozoite invasion of red blood cells in vitro. No variation in sensitivity to heparin was found among the four strains of P. falciparum tested; all required approximately 5 micrograms/ml (0.5 U/ml) of heparin for 50% inhibition of invasion. The most efficient fraction of heparin was the one with low affinity for antithrombin III. Its 50% inhibition concentration was 1 microgram/ml, indicating that it was more efficient than unfractionated heparin and other heparin fractions. The effect of heparin was reversible, since washing of heparin-treated cultures containing mainly schizonts showed no inhibition of merozoite invasion. The results suggest that a heparin fraction with no anticoagulant effect might be useful in the treatment of patients with falciparum malaria.

In vitro inhibition of Plasmodium falciparum merozoite invasion by human plasma fibronectin.

Human fibronectin isolated from citrated blood was tested for its ability to bind to Plasmodium falciparum by an indirect immunofluorescent assay using rabbit antiserum to human fibronectin. A positive reaction was observed on merozoites inside schizont-infected erythrocytes. The binding was not observed on non-parasitized red blood cells. The effect of human fibronectin on P. falciparum growth was further studied using an in vitro inhibition assay; 50% inhibition of parasite multiplication was obtained with approximately 100 micrograms/ml of human fibronectin. Slight inhibition was observed below 10 micrograms/ml. The significance of this finding is discussed.

Effects of Neoglycoproteins on Penetration of Plasmodium falciparum Merozoites into Erythrocytes In vitro

Sugars conjugated to bovine serum albumin (neoglycoproteins) were tested for their ability to inhibit Plasmodium falciparum merozoite invasion of red blood cells in an in vitro inhibition assay. The inhibitory effects of the neoglycoproteins on merozoite invasion of erythrocytes were determined by assessing 3H-hypoxanthine incorporation into the cultures. The only neoglycoprotein that caused significant inhibition N-acetyl-glucosamine-bovine serum albumin. A comparison of the inhibitory effects of N-acetyl-glucosamine and N-acetyl-glucosamine-bovine serum albumin showed that N-acetyl-glucosamine-bovine serum albumin was 84 fold more effective than the free sugar in causing 50% inhibition of 3H-hypoxanthine incorporation.

Assay of erythrocyte components as inhibitors of Plasmodium falciparum merozoite invasion of erythrocytes.

Three red blood cell membrane fractions (Band 3, macromolecules exhibiting I antigenic determinants, and a delipidated glycoprotein fraction) were separated from red blood cell membranes and tested for their ability to inhibit penetration of red blood cells by Plasmodium falciparum merozoites in an in vitro inhibition assay. The delipidated glycoprotein fraction (containing the major sialoglycoproteins and devoid of Band 3) was the only fraction that inhibited merozoite invasion. This fraction showed 73% and 70% inhibition at 1 mg/ml and 500 micrograms/ml, respectively, and slight inhibition below these levels.

Plasmodium falciparum: Assay of invasion of erythrocytes

A method for quantitatively assaying Plasmodium falciparum merozoite invasion of particular erythrocytes is described. Erythrocytes were labeled with fluorescein isothiocyanate which did not affect parasite entry or growth, to distinguish them from uninfected erythrocytes in the original parasitized cell population. Parasites were detectable after staining with ethidium bromide. The time course of infection of the labeled cells was followed over 26 hr. The technique was used to determine the effect of serum from a patient with P. falciparum malaria on merozoite invasion of the labeled erythrocytes.