Gametocyte formation by the progeny of single Plasmodium falciparum schizonts

Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.     

A developmental defect in Plasmodium falciparum male gametogenesis

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha- tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.     

Differential adhesive properties of sequestered asexual and sexual stages of Plasmodium falciparum on human endothelial cells are tissue independent

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.     

Plasmodium falciparum: mRNA co-expression and protein co-localisation of two gene products upregulated in early gametocytes

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.     

Gametocyte clearance in uncomplicated and severe Plasmodium falciparum malaria after artesunate-mefloquine treatment in Thailand

Artemisinin-based combination therapy (ACT) is currently promoted as a strategy for treating both uncomplicated and severe falciparum malaria, targeting asexual blood-stage Plasmodium falciparum parasites. However, the effect of ACT on sexual-stage parasites remains controversial. To determine the clearance of sexual-stage P. falciparum parasites from 342 uncomplicated, and 217 severe, adult malaria cases, we reviewed and followed peripheral blood sexual-stage parasites for 4 wk after starting ACT. All patients presented with both asexual and sexual stage parasites on admission, and were treated with artesunate-mefloquine as the standard regimen. The results showed that all patients were asymptomatic and negative for asexual forms before discharge from hospital. The percentages of uncomplicated malaria patients positive for gametocytes on days 3, 7, 14, 21, and 28 were 41.5, 13.1, 3.8, 2.0, and 2.0%, while the percentages of gametocyte positive severe malaria patients on days 3, 7, 14, 21, and 28 were 33.6, 8.2, 2.7, 0.9, and 0.9%, respectively. Although all patients were negative for asexual parasites by day 7 after completion of the artesunate-mefloquine course, gametocytemia persisted in some patients. Thus, a gametocytocidal drug, e.g., primaquine, may be useful in combination with an artesunate-mefloquine regimen to clear gametocytes, so blocking transmission more effectively than artesunate alone, in malaria transmission areas.     

Structure and non-essential function of glycerol kinase in Plasmodium falciparum blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito.     

Stage-specific inhibitory effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum

The effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum grown in vitro was examined over a wide range of concentrations. Cyclic AMP inhibited both processes in a stage-specific manner. Asexual maturation was inhibited from shortly after parasite entry into the red cell through the ring stage. However, trophozoites and schizonts matured normally in the presence of cyclic AMP and produced infectious merozoites. Gametocyte formation was inhibited by 95% when 1.0 mM cyclic AMP was added to synchronously growing parasites in the ring stage of development but was only inhibited by 15% when added in the trophozoite or schizont stages. Cyclic AMP was not found to increase gametocyte formation over a wide range of concentrations.     

The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion

Much of the epidemiology of Plasmodium falciparum in Sub-Saharan Africa focuses on the prevalence patterns of asexual parasites in people of different ages, whereas the gametocytes that propagate the disease are often neglected. One expected benefit of the widespread introduction of artemisinin-based combination therapy for malaria is a reduction in gametocyte carriage. However, the factors that affect the transmission of parasites from humans to mosquitoes show complex dynamics in relation to the intensity and seasonality of malaria transmission, and thus such benefits might not be automatic. Here, we review data on gametocyte carriage in the context of the development of naturally acquired immunity and population infectivity.     

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood-feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage-specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue-specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.     

Differentiation of Gametocytes in Microcultures of Human Blood Infected with Plasmodium falciparum*

SYNOPSIS. Gametocytes differentiated from ring-stage parasites in microcultures of human blood infected with Plasmodium falciparum. Immature gametocytes could be distinguished morphologically from late asexual trophozoites after ～ 40 h of culture. Differentiation into crescentic forms took several days and the incorporation of [³H]-isoleucine by developing gametocytes was demonstrated. About 1% of red cells contained gametocytes at the maximum densities attained. Differentiation of gametocytes occurred either directly from rings placed in culture or from the progeny of subsequent cycles of schizogony and invasion in vitro. The latter occurrence was confirmed by the development of gametocytes in marker fetal red cells added to cultures, although fetal red cells provide a less favorable environment than those with HbA for growth of the parasites.     

Decreased hemoglobin concentrations,hyperparasitemia, and severe malaria are associated with increased Plasmodium falciparum gametocyte carriage

To determine factors influencing gametocyte carriage, a cross-sectional study was conducted among 512 patients admitted for Plasmodium falciparum malaria. After adjustments for potential confounders, hemoglobin concentrations were lower in gametocyte carriers 10.5 (+/-2.5) than in patients without gametocytes 12.5 (+/-2.3) (P < 0.0001). Hemoglobin concentrations were negatively correlated with peak gametocyte counts (Spearman's p = -0.37, P < 0.0001) and gametocyte carriage durations (Spearman's p = -(0.30, P < 0.0001). Adjustments for the duration of the malaria episode and other potential confounders did not alter the association (P < 0.0001). After adjustment for potential confounders, the median asexual parasitemia was higher in patients with gametocytes than in patients without gametocytes (P = 0.003). Severe malaria cases were more likely to have gametocytes (65%) than malaria with hyperparasitemia (38%) or mild malaria (31%) (P = 0.0001). These findings suggest that events surrounding anemia and tissue hypoxia stimulate Plasmodium falciparum gametocytogenesis.     

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.     

Appropriate Time for Primaquine Treatment to Reduce Plasmodium falciparum Transmission in Hypoendemic Areas

Artemesinin-combination therapies (ACT) for falciparum malaria reduce gametocyte carriage, and therefore reduce transmission. Artemisinin derivatives will act against only young gametocytes whereas primaquine acts on mature gametocytes which are present usually in the circulation at the time when the patient presents for treatment. Both artemisinin derivatives and primaquine have short half-lives, less than 1 hr and 7 hr, respectively. Therefore, asexual parasites or young gametocytes remain after completed ACT. A single dose of primaquine (0.50-0.75 mg base/kg) at the end of ACT can kill only mature gametocytes but cannot kill young gametocytes (if present). Remaining asexual forms after completion of ACT course, e.g., artesunate-mefloquine for 3 days, may develop to mature gametocytes 7-15 days later. Thus, an additional dose of primaquine (0.50-0.75 mg base/kg) given 2 weeks after ACT completion may be beneficial for killing remaining mature gametocytes and contribute to more interruption of Plasmodium falciparum transmission than giving only 1 single dose of primaquine just after completing ACT.     

The effect of partial host immunity on the transmission of malaria parasites.

Experiments were carried out to determine the effect of partial host immunity against the rodent malaria parasite Plasmodium chabaudi on the transmission success of the parasite. There was a fourfold reduction in both the blood-stage, asexually replicating parasite density and the gametocyte (transmissable stage) density in immunized hosts. Some of the reduction in asexual parasite densities was due to strain-specific immunity, but there was no evidence that strain-specific immunity affected gametocyte densities. However, immunity did affect transmission in a strain-specific manner, with a fivefold reduction in gametocyte infectivity to mosquitoes in homologous challenges compared with heterologous challenges or non-immunized controls. This implies the existence of a mechanism of strain-specific infectivity-reducing immunity that does not affect the density of gametocytes circulating in peripheral blood. The proportion of asexual parasites that produced gametocytes increased during the course of infection in both non-immunized and in immunized hosts, but immunity increased gametocyte production early in the infection.     

A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.     

Isotype expression, post-translational modification and stage-dependent production of tubulins in erythrocytic Plasmodium falciparum

Microtubules are cytoskeletal polymers containing repeating alpha/beta-tubulin heterodimers and are found in all eukaryotes including the malaria parasite Plasmodium falciparum. Diverse cellular functions such as chromosomal segregation, organelle transport and the determination of cell shape and motility are all dependent on microtubules. This essential role played by tubulin in cells is reflected in the effective use of anti-microtubule agents as fungicides, herbicides, anti-parasitic and anti-cancer agents. Plasmodium falciparum microtubules have been proposed as a potential antimalarial drug target and knowledge of their molecular composition and cellular architecture in blood-stage parasites is required to substantiate this premise. We report here that: (i) the two alpha-tubulin isotypes, alphaI- and alphaII-tubulin, are produced in both asexual and sexual blood-stage parasites, contrary to the previous report that alphaII-tubulin was specific to male gametocytes; (ii) tubulin production is highly stage-dependent in asexual parasites, reaching its maximum level in schizonts and segmenters and (iii) there is evidence of post-translational polyglutamylation of tubulin. The glutamylation of P. falciparum tubulins is the first reported post-translational modification of tubulin in this organism and was found only in the microtubule-organising centres and post-mitotic microtubular structures, suggesting possible roles for this modification in spindle pole body formation and merozoite biogenesis. Taken together, these findings form the basis for a better biological appreciation of P. falciparum microtubules and for the correct deployment of purified tubulins in the evaluation of microtubule inhibitors as potential antimalarial drugs.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.