Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.     

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.     

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.     

Relationship between partial inhibition of glycolysis and hemolysis after induction of gametocytogenesis in synchronous cultures of Plasmodium falciparum

In the present study, the low molecular-weight fraction of the culture supernatant of anti-Plasmodium falciparum antibody-producing hybridoma cells (HybSL) was used in synchronous culture with P. falciparum FVO strain. When synchronous cultures were treated with HybSL solution on day 5, gametocytogenesis was also induced. Gametocytes were consistently found from the third day after treatment and reached a peak on the fourth day. An increase in pH and hemoglobin concentrations and decrease in lactate concentrations were observed on the first day after treatment. These phenomena suggested that HybSL solution partially inhibited glycolysis of erythrocytes parasitized with schizonts and resulted in hemolysis of infected erythrocytes. On the other hand, the production of gametocytes did not increase in cultures treated with HybSL solution on day 4 of synchronous cultures in which ring forms were plentiful. Most ring forms were not killed by HybSL solution and quickly developed to trophozoites and schizonts rather than gametocytes. Consequently, it is assumed that ring forms on day 4 of synchronous cultures have finished differentiation into the asexual stage. The conversion of asexual parasites to gametocytes may be triggered only when late-stage trophozoites or early-stage schizonts are treated with HybSL solution.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Falciparum malaria in naturally infected human patients: II. Ultrastructural alterations to erythrocytes infected with asexual forms

Ultrastructural alterations of human erythrocytes infected with asexual forms of Plasmodium falciparum were studied in naturally infected Saudi patients. These included surface knobs and nodules as well as invaginations associated with cytopasmic vesicles observed in erythrocytes infected with asexual forms of the parasites. Such nodules and surface invaginations have been previously described only in erythrocytes infected with P. ovale and P. vivax, respectively. Within the cytoplasm of infected erythrocytes were membrane-bound clefts, similar to those that appear to be a common characteristic in all red cells infected with malaria parasites. Vacuolations were often seen in the peripheral cytoplasm and may represent hemolyzed areas. Collapsed cells with an internal-lucent interior and surrounded by an irregularly folded membrane may represent completely hemolyzed erythrocytes. © 1993 Wiley-Liss, Inc.     

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.     

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.     

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.     

Asexual and Sexual Stages of a Malaria Parasite in the Thrombocytes of Tropidurus torquatus (Iguanidae) Infected with Plasmodium tropiduri*

SYNOPSIS. Sexual and asexual stages of a parasite found in the thrombocyte-like cells of some Tropidurus torquatus infected with Plasmodium tropiduri are described. The strong ultrastructural similarities between the gametocytes of this parasite and the gametocytes of P. tropiduri, and the finding of this parasite in lizards inoculated with P. tropiduri suggest that this malaria parasite can develop both in erythrocytes and thrombocytes. Evidence in favor of this hypothesis is discussed.     

Malaria infection increases attractiveness of humans to mosquitoes

Do malaria parasites enhance the attractiveness of humans to the parasite's vector? As such manipulation would have important implications for the epidemiology of the disease, the question has been debated for many years. To investigate the issue in a semi-natural situation, we assayed the attractiveness of 12 groups of three western Kenyan children to the main African malaria vector, the mosquito Anopheles gambiae. In each group, one child was uninfected, one was naturally infected with the asexual (non-infective) stage of Plasmodium falciparum, and one harboured the parasite's gametocytes (the stage transmissible to mosquitoes). The children harbouring gametocytes attracted about twice as many mosquitoes as the two other classes of children. In a second assay of the same children, when the parasites had been cleared with anti-malarial treatment, the attractiveness was similar between the three classes of children. In particular, the children who had previously harboured gametocytes, but had now cleared the parasite, were not more attractive than other children. This ruled out the possibility of a bias due to differential intrinsic attractiveness of the children to mosquitoes and strongly suggests that gametocytes increase the attractiveness of the children.     

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.     

Gametocyte development of Plasmodium chabaudi in mice and rats: evidence for host induction of gametocytogenesis

Gametocytogenesis in Plasmodium chabaudi was studied in intact and splenectomized mice and rats. Blood forms inoculated into mice entered a new asexual cycle, resulting in high parasitaemias 4 days after inoculation. Blood forms inoculated into intact rats were almost eliminated by day 4 after injection. In splenectomized rats, however, the parasitaemia remained constant over this period. With an immunofluorescence test (IFA), using an antiserum to mouse erythrocytes, it was found that the majority of parasites invaded rat erythrocytes, but were unable to enter a new asexual cycle. It was found that up to 50% of the parasites in splenectomized rats developed into gametocytes. The IFA showed that the proportion of gametocytes to asexual forms was very high in splenectomized animals, irrespective of the origin of the host cells-mouse or rat.     

A novel Plasmodium yoelii pseudokinase,PypPK1, is involved in erythrocyte invasion and exflagellation center formation

Malaria parasites proliferate by repeated invasion of and multiplication within erythrocytes in the vertebrate host. Sexually committed intraerythrocytic parasites undergo sexual stage differentiation to become gametocytes. After ingestion by the mosquito, male and female gametocytes egress from erythrocytes and fertilize within the mosquito midgut. A complex signaling pathway likely responds to environmental events to trigger gametogenesis and regulate fertilization; however, such knowledge remains limited for malaria parasites. Several pseudokinases are highly transcribed at the gametocyte stage and are possible multi-functional regulators controlling critical steps of the life cycle. Here we characterized one pseudokinase, termed PypPK1, in Plasmodium yoelii that is highly expressed in schizonts and male gametocytes. Immunofluorescence assays for parasites expressing Myc-tagged PypPK1 confirmed that PypPK1 protein is expressed in schizonts and sexual stage parasites. Transgenic ΔpPK1 parasites, in which the PypPK1 gene locus was deleted by the CRISPR/Cas9 method, showed significant growth defect and reduced virulence in mice. In the blood stage, ΔpPK1 parasites were able to egress from erythrocytes similar to wild type parasites; however, erythrocyte invasion efficacy was significantly reduced. During sexual stage development, no clear changes were seen in male and female gametocytemias as well as gametocyte egress from erythrocytes; but, the number of exflagellation centers and oocysts were significantly reduced in ΔpPK1 parasites. Taken together, PypPK1 has an important role for both erythrocyte invasion and exflagellation center formation.     

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.     

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.     

Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes – the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue , is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Δ mdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.