Plasmodium falciparum gametocytes: their longevity and infectivity.

The longevity and infectivity of isolated populations of Plasmodium falciparum gametocytes were studied. Following chloroquine treatment gametocyte numbers fell with a constant rate of loss over a period of 16-24 days; the populations had a half-life of 2-4 days. The sex ratio stayed constant throughout at 4 female: 1 male. The ability of the microgametocytes to exflagellate and the infectivity of the population to mosquitoes persisted for 3 weeks. Antibodies to the gametocytes were detected but not in every patient studied. It was concluded that the gametocytes of P. falciparum are both long-lived and show persistent infectivity to mosquitoes. They can stimulate antibody production but the immune response appears to play no part in their elimination, which probably takes place in the spleen as a part of the normal process of removing old, damaged and malformed red cells.     

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.     

Plasmodium falciparum: induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro

A genetically homogeneous population of Plasmodium falciparum prepared by a single erythrocyte micromanipulation technique was used to produce lines of P. falciparum resistant to mefloquine hydrochloride in vitro. Parasites were maintained in a culture medium containing gradually increased concentrations of mefloquine hydrochloride (CMP-mef) starting with 2 ng/ml. One of the mefloquine-resistant culture lines (W2-mef) was obtained after 96 weeks of continuous culture in CMP-mef, the last 4 weeks in medium containing 40 ng/ml of mefloquine hydrochloride. The W2-mef was four to six times more resistant to mefloquine than was the parent clone W2. Means of multiple determinations of 50% inhibitory concentrations (IC-50) of mefloquine hydrochloride against W2-mef and clone W2 were 20.39 +/- 5.08 ng/ml and 4.50 +/- 1.94 ng/ml, respectively.     

Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.

Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.     

In vitro posttranslational modification of lamin B cloned from a human T-cell line.

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.     

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Specific detection of Plasmodium falciparum malaria by a molecularly cloned DNA probe

A highly repeated DNA sequence from Plasmodium falciparum was cloned and used as a probe in molecular hybridization to detect malaria. Our results indicate that the probe is specific to P. falciparum but not to other species of Plasmodium and is extremely sensitive. As little as a 20 pg parasite DNA, which is equivalent to about 1000 parasites can be detected. The cloned DNA can be used as a diagnostic tool to follow the course of infection of falciparum malaria.     

High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.     

Effect of xanthurenic acid on infectivity of Plasmodium falciparum to Anopheles stephensi.

Terminally differentiated malarial gametocytes remain in the vertebrate circulation in a developmentally arrested state until they are taken up by the mosquito. The gametocytes then undergo gametogenesis in the mosquito mid-gut within minutes after ingestion of the infected blood meal. The male gametogenesis (exflagellation) can be triggered by the combination of a decrease in temperature of at least 5 degrees C and a simultaneous increase in pH between 8.0 and 8.3. Xanthurenic acid, which is present in mosquito mid-gut as well as in mosquito head, had been shown to induce exflagellation in vitro at a non-permissible pH. Here we report for the first time that with the increasing concentration of exogenous xanthurenic acid, there is a gradual increase in the number of oocysts in the mid-gut of infected mosquitoes. The concentration of xanthurenic acid for optimum infection in the membrane feeding assay was determined to be 100 microM. Three different strains of Plasmodium falciparum, viz. 3D7, 7G8 and W2 were tested in different experiments and similar findings hold true for all of them. These results demonstrate that xanthurenic acid not only induces exflagellation of male gametocytes but also promotes infectivity of Plasmodium falciparum to mosquito vectors.     

Plasmodium falciparum: isolation and characterization of a 55-kDa protease with a cathepsin D-like activity from P. falciparum

Native electrophoresis followed by imprint digest method using hemoglobin as substrate allowed the detection of parasite hemoglobinase activity at acidic pH (3.9 to 5). This protease was inhibited specifically by pepstatin A and insensitive to other protease inhibitors. The molecular weight determination using modified SDS-PAGE followed by imprint digest method, demonstrated a single area of activity at 55-58 kDa, similar to cathepsin D characterized in eucaryotic cells. The parasitic origin has been shown by radiolabeling experiments with [35S]-methionine. The 55-kDa protein was immunoprecipitated by a rabbit anti-cathepsin D serum.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

Expression of cloned cDNA for a major surface antigen of Plasmodium falciparum merozoites

A cDNA library of P. falciparum was constructed. Using size-selected mRNA as a probe several clones were isolated which hybridized to mRNAs larger than 5 kilobases (kb). The cDNA insert of pFC 17, which hybridizes to 5.6-kb mRNA was expressed by fusion to anthranilate synthetase I in a plasmid expression vector. The expressed fusion protein was shown to contain epitopes of a 195-kDa protein which is the precursor to 3 major surface antigens of P. falciparum merozoites.     

Plasmodium falciparum: merozoite invasion in vitro in the presence of chloroquine.

The fine structure of invasion of human erythrocytes by merozoites of the malaria parasite Plasmodium falciparum was observed in vitro. The invasion process is similar to that described for P. knowlesi. Merozoites enter apical end first by invagination of the erythrocyte membrane. At the rim of the invagination, where merozoite and erythrocyte are in closest contact, the erythrocyte membrane is thickened. The brushy cell coat of the P. falciparum merozoite appears to be lost at this attachment zone. The part of the merozoite within the erythrocyte invagination has no visible coat. The coat on the portion outside is unaltered. Merozoites can successfully invade erythrocytes after 3 hr in the presence of a concentration of chloroquine harmful to feeding stages.     

Plasmodium falciparum: microaerophilic requirements in human red blood cells.

The respiratory requirements of Plasmodium falciparum were studied in vitro in continuous cultivation. The cultures were held in petri dishes containing the parasites incubated in different gas mixtures for periods of 72 to 144 hr with daily media changes. Atmospheres were combinations of 0.5 to 21% O₂ mixed with 1 to 5% CO₂ diluted with N₂. Gas concentrations and the pH of media were measured with an $\text{O}{}_2\text{CO}{}_2$ analyzer. Best growth was realized in all cases at 3% O₂ and 1 to 2% CO₂. The culture appeared to be selfperpetuating in O₂ concentrations as low as 0.5% providing the CO₂ was not over 2%. Oxygen concentrations of 21% proved deleterious to growth. The parasite however, failed to grow in the highly reducing atmosphere of anaerobic “Brewer Jars,” suggesting that P. falciparum is an obligate microaerophile.     

Plasmodium falciparum: in vitro induction of resistance to aminopterin

Plasmodium falciparum parasites were grown on microplates in the presence of aminopterin. The FCR-8 strain was more sensitive to aminopterin than a Richards strain and died within 1 week of treatment. A few parasites of the Richards strain survived treatment and developed normal parasitemias. This strain was resistant to aminopterin at concentrations not higher than those used for its selection. Removal of aminopterin did not affect the growth of the resistant variant, showing that it was not aminopterin dependent. Aminopterin affected the sensitive parasites by interfering with nucleic acid synthesis, whereas protein synthesis was not impaired. Gametocytogenesis was unaffected by aminopterin.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.