Cyclosporin-binding proteins of Plasmodium falciparum

A number of cyclosporins, including certain non-immunosuppressive ones, are potent inhibitors of the intraerythrocytic growth of the human malarial parasite Plasmodium falciparum. The major cyclosporin-binding proteins of P. falciparum were investigated by affinity chromatography on cyclosporin-Affigel followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting, and peptide mass fingerprinting. The two bands obtained on gels were shown to correspond to cyclophilins, PfCyP-19A (formerly PfCyP-19) and PfCyP-19B, whose genes had been characterised previously. PfCyP-19B was an abundant protein of intraerythrocytic P. falciparum (up to 0.5% of parasite protein) that was present in the highest amounts in schizont-stage parasites. Unexpectedly, given its apparent signal sequence, it was located primarily in the cytosol of the parasite. The peptidyl-prolyl cis-trans isomerase activity of recombinant PfCyP-19B had the same profile of susceptibility to cyclosporin derivatives as the bulk isomerase activity of crude P. falciparum extracts. The binding of cyclosporins to cyclophilins may be relevant to the mechanism of action of the drug in the parasite.     

Plasmodium falciparum: growth response to potassium channel blocking compounds

Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K⁺ channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca²⁺-activated K⁺ channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K⁺ channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca²⁺-activated K⁺ channel blocking compounds.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum

A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.     

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.     

The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15-26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II-IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

The role of tryptophan-48 in catalysis and binding of inhibitors of Plasmodium falciparum dihydrofolate reductase

Dihydrofolate reductases (DHFRs) from Plasmodium falciparum (Pf) and various species of both prokaryotic and eukaryotic organisms have a conserved tryptophan (Trp) at position 48 in the active site. The role in catalysis and binding of inhibitors of the conserved Trp48 of PfDHFR has been analysed by site-specific mutagenesis, enzyme kinetics and use of a bacterial surrogate system. All 19 mutant enzymes showed undetectable or very low specific activities, with the highest value of k(cat)/K(m) from the Tyr48 (W48Y) mutant (0.12 versus 11.94M(-1)s(-1)), of about 1% of the wild-type enzyme. The inhibition constants for pyrimethamine, cycloguanil and WR99210 of the W48Y mutants are 2.5-5.3 times those of the wild-type enzyme. All mutants, except W48Y, failed to support the growth of Escherichia coli transformed with the parasite gene in the presence of trimethoprim, indicating the loss of functional activity of the parasite enzyme. Hence, Trp48 plays a crucial role in catalysis and inhibitor binding of PfDHFR. Interestingly, W48Y with an additional mutation at Asn188Tyr (N188Y) was found to promote bacterial growth and yielded a higher amount of purified enzyme. However, the kinetic parameters of the purified W48Y+N188Y enzyme were comparable with W48Y and the binding affinities for DHFR inhibitors were also similar to the wild-type enzyme. Due to its conserved nature, Trp48 of PfDHFR is a potential site for interaction with antimalarial inhibitors which would not be compromised by its mutations.     

Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.     

A modified Plasmodium falciparum growth inhibition assay (GIA) to assess activity of plasma from malaria endemic areas

Plasma samples from patients undergoing treatment in malaria endemic countries often contain anti-malaria drugs, that may overstate effects of specific antibodies in growth inhibition assays (GIA). We describe a modified assay that uses drug resistant P. falciparum parasites (W2) that circumvents the requirement for dialyzing samples that may likely contain drugs such as chloroquine and sulfadoxine/pyrimethamine (SP).     

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

Inhibition of Plasmodium falciparum proliferation in vitro by antisense oligodeoxynucleotides against malarial topoisomerase II

The development of new effective antimalarial agents is urgently needed due to the ineffectiveness of current drug regimes on the most virulent human malaria parasite Plasmodium falciparum. Antisense (AS) oligodeoxynucleotides (ODNs) have shown promise as chemotherapeutic agents. Phosphorothioate AS ODNs against different regions of P. falciparum topoisomerase II gene were investigated. Chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was exposed to phosphorothioate AS ODNs for 48 h and growth was determined by flow cytometric assay or by microscopic assay. Exogenous delivery of phosphorothioate AS ODNs between 0.01 and 0.5 microM significantly inhibited parasite growth compared with sense sequence controls suggesting sequence specific inhibition. This inhibition was shown to occur during maturation stages, with optimal inhibition being detected after 36 h. These results should prove useful in future designs of novel antimalarial agents.     

Conflicting immune responses can prolong the length of infection in Plasmodium falciparum malaria

We have recently proposed a new model for antigenic variation in Plasmodium falciparum that relies on a network of partially cross-protective immune responses to orchestrate this complex immune evasion process. In addition to exhibiting prolonged oscillations of single variants that resemble the sequential dominance of immunologically distinct antigenic types, the model implies that a higher efficacy of cross-reactive immunity actually increases the length of infection while reducing severity of disease. Here, we analyse the behaviour of a reduced system under conditions of perfect synchrony between variants to demonstrate that these features of this system can be attributed to the antagonism between cross-reactive and variant-specific responses.     

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.     

Delayed parasite elimination in human infections treated with clindamycin parallels 'delayed death' of Plasmodium falciparum in vitro

Clindamycin is safe and effective for the treatment of Plasmodium falciparum malaria, but its use as monotherapy is limited by unacceptably slow initial clinical response rates. To investigate whether the protracted action is due to an accumulative, time of exposure-dependent or a delayed effect on parasite growth, we studied the in vivo and in vitro pharmacodynamic profiles of clindamycin against P. falciparum. In vivo, elimination of young, circulating asexual parasite stages during treatment with clindamycin displayed an unusual biphasic kinetic: a plateau phase was followed by a precipitated decline of asexual parasite densities to nearly undetectable levels after 72 and 60 h in adult patients and asymptomatic children, respectively, suggesting an uninhibited capacity to establish a second, but not third, infectious cycle. In vitro, continuous exposure of a laboratory-adapted P. falciparum strain to clindamycin with concentrations of up to 100 microM for two replication cycles (96 h) did not produce inhibitory effects of >50% compared with drug-free controls as measured by the production of P. falciparum histidine-rich protein II (PfHRP2). PfHRP2 production was completely arrested after the second cycle (96-144h) (>10,000-fold decrease of mean half-inhibitory concentrations measured at 96-144h compared to 48-96h). Furthermore, incubation with clindamycin during only the first (0-48h) versus three (0-144h) parasite replication cycles led to comparable inhibition of PfHRP2 production in the third infectious cycle (96-144h) (mean IC(99) of 27 and 22nM, respectively; P=0.2). When parasite cultures were exposed to different concentrations of clindamycin ranging from 50 to 1,000nM for 72h and followed up in an experiment designed to simulate a typical 3-day treatment regimen, parasitaemia was initially suppressed below the microscopic detection threshold. Nonetheless, parasites reappeared in a dose-dependent manner after removal of drug at 72h but not in continuously drug-exposed controls. The delayed, but potent, antimalarial effect of clindamycin appears to be of greatest potential benefit in new combinations of clindamycin with rapidly acting antimalarial combination partners.     

The human placental derived BeWo cell line: a useful model for selecting Plasmodium falciparum CSA-binding parasites

Chondroitin sulfate A (CSA) present in the placental intervillous blood spaces has been described as the main receptor involved in the massive sequestration of Plasmodium falciparum parasitized erythrocytes to the placenta. Placental parasite isolates are functionally distinct from isolates that sequester in other organs, because they do not cytoadhere to CD36 but instead bind to CSA. To investigate for the parasites molecules associated with the CSA adhesion phenotype, different methodologies have been developed to select for CSA-binding lines in vitro mainly using non-placental sources of CSA that differ in their sulfation pattern. In this study, we show that the human trophoblastic BeWo cell line is a very efficient alternative to select for the CSA-binding phenotype in parasitized erythrocytes.     

Plasmodium yoelii: experimental evidences for the conserved epitopes between mouse and human malaria parasite, Plasmodium falciparum

Bioinformatic analyses of gene homologues have revealed functionally conserved epitopes between human and rodent malaria parasites. Here, we present experimental evidence for the presence of functionally and antigenically conserved domains between Plasmodium falciparum and Plasmodium yoelii asexual blood-stages. Merozoite released soluble proteins (MRSPs) from both P. falciparum and P. yoelii bound to heterologous mouse or human red blood cells, respectively. The presence of conserved antigenic epitopes between the two species of parasites was evident by the inhibitory effect of antibodies, developed against P. yoelii in convalescent mice, on P. falciparum growth and merozoite reinvasion in vitro. Furthermore, mice immunized with P. falciparum MRSPs were protected from infection by a P. yoelii challenge. These data indicate that different species of Plasmodium contain antigenically conserved interspecies domains, which are immunogenic and, thus constitute a potential novel antigen source for vaccine development and testing using a mouse model.