scholarly journals Novel Method for the Separation of Male and Female Gametocytes of the Malaria Parasite Plasmodium falciparum That Enables Biological and Drug Discovery

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Melanie C. Ridgway ◽  
Kwong Sum Shea ◽  
Daniela Cihalova ◽  
Alexander G. Maier
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Malaria Transmission ◽  
Disease Transmission ◽  
Severe Form ◽  
Male And Female ◽  
Content Type ◽  
Single Sex ◽  
Parasite Plasmodium Falciparum ◽  
Female Bias

ABSTRACT We developed a flow-cytometry-based method to separate and collect cocultured male and female Plasmodium falciparum gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific P. falciparum gametocyte biology to further our understanding of malaria transmission. IMPORTANCE The protozoan Plasmodium falciparum causes the most severe form of human malaria. The development of sexual forms (so-called gametocytes) is crucial for disease transmission. However, knowledge of these forms is severely hampered by the paucity of sex-specific markers and the inability to extract single sex gametocytes in high purity. Moreover, the identification of compounds that specifically affect one sex is difficult due to the female bias of the gametocytes. We have developed a system that allows for the separation of male and female gametocytes from the same population. Applying our system, we show that male and female parasites mature at different rates, which might have implications for transmission. We also identified new sex-specific genes that can be used as sex markers or to unravel sex-specific functions. Our system will not only aid in the discovery of much needed gametocidal compounds, but it also represents a valuable tool for exploring malaria transmission biology.

scholarly journals Seroprevalence of Antibodies against Plasmodium falciparum Sporozoite Antigens as Predictive Disease Transmission Markers in an Area of Ghana with Seasonal Malaria Transmission

PLoS ONE ◽  
2016 ◽  
Vol 11 (11) ◽  
pp. e0167175 ◽  
Author(s):  
Kwadwo A. Kusi ◽  
Samuel Bosomprah ◽  
Eric Kyei-Baafour ◽  
Emmanuel K. Dickson ◽  
Bernard Tornyigah ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Transmission ◽  
Disease Transmission ◽  
Falciparum Sporozoite

Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum

Parasitology ◽  
1999 ◽  
Vol 119 (1) ◽  
pp. 7-17 ◽  
Author(s):  
H. A. GIHA ◽  
T. STAALSOE ◽  
D. DODOO ◽  
I. M. ELHASSAN ◽  
C. ROPER ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Malaria Transmission ◽  
Erythrocyte Membrane ◽  
Clonal Variation ◽  
Single Infection ◽  
Erythrocyte Membrane Protein ◽  
Degree Of Overlap ◽  
Eastern Sudan

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

scholarly journals Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and FemalePlasmodium falciparumGametocytes

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Katelyn A. Walzer ◽  
Danielle M. Kubicki ◽  
Xiaohu Tang ◽  
Jen-Tsan Ashley Chi
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Single Cell ◽  
Specific Expression ◽  
Male And Female ◽  
Content Type ◽  
Population Analyses ◽  
Specific Differentiation ◽  
Female Specific ◽  
Gender Specific

ABSTRACTSexual reproduction is an obligate step in thePlasmodium falciparumlife cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought to be specific to gametocytes, only a few have been postulated to be male or female specific. Because these genes are often expressed during late gametocyte stages and for some, male- or female-specific transcript expression is debated, the separation of male and female populations is technically challenging. To overcome these challenges, we have developed an unbiased single-cell approach to determine which transcripts are expressed in male versus female gametocytes. Using microfluidic technology, we isolated single mid- to late-stage gametocytes to compare the expression of 91 genes, including 87 gametocyte-specific genes, in 90 cells. Such analysis identified distinct gene clusters whose expression was associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. Many female-enriched genes also exhibited stage-specific expression. RNA fluorescentin situhybridization of male and female markers validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight intoPlasmodiumsex-specific differentiation previously masked in population analyses. Our single-cell approach reveals the most robust markers for sex-specific differentiation inPlasmodiumgametocytes. Such single-cell expression assays can be generalized to all eukaryotic pathogens.IMPORTANCEMost human deaths that result from malaria are caused by the eukaryotic parasitePlasmodium falciparum. The only form of this parasite that is transmitted to the mosquito is the sexual form, called the gametocyte. The production of mature gametocytes can take up to 2 weeks and results in phenotypically distinct males and females, although what causes this gender-specific differentiation remains largely unknown. Here, we demonstrate the first use of microfluidic technology to capture single gametocytes and determine their temporal sex-specific gene expression in an unbiased manner. We were able to determine male or female identity of single cells based on the upregulation of gender-specific genes as early as mid-stage gametocytes. This analysis has revealed strong markers for male and female gametocyte differentiation that were previously concealed in population analyses. Similar single-cell analyses in eukaryotic pathogens using this method may uncover rare cell types and heterogeneity previously masked in population studies.

scholarly journals A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

2013 ◽  
Vol 12 (9) ◽  
pp. 1179-1191 ◽  
Author(s):  
Sebastian Fernandez-Pol ◽  
Zdenek Slouka ◽  
Souvik Bhattacharjee ◽  
Yana Fedotova ◽  
Stefan Freed ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Phosphatase Activity ◽  
Tyrosine Phosphatase ◽  
Protein Band ◽  
Band 3 ◽  
Dense Granule ◽  
Content Type ◽  
Cytoskeletal Network ◽  
Parasite Plasmodium Falciparum

ABSTRACT Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella -like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro , PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.

scholarly journals Antimalarial Iron Chelator FBS0701 Blocks Transmission by Plasmodium falciparum Gametocyte Activation Inhibition

2014 ◽  
Vol 59 (3) ◽  
pp. 1418-1426 ◽  
Author(s):  
Patricia Ferrer ◽  
Joel Vega-Rodriguez ◽  
Abhai K. Tripathi ◽  
Marcelo Jacobs-Lorena ◽  
David J. Sullivan
Keyword(s):  
Plasmodium Falciparum ◽  
Ferric Chloride ◽  
Iron Metabolism ◽  
Iron Chelator ◽  
Inhibitory Effect ◽  
Malarial Parasite ◽  
Iron Chelators ◽  
Simultaneous Administration ◽  
Male And Female ◽  
Content Type

ABSTRACTReducing the transmission of the malarial parasite byAnophelesmosquitoes using drugs or vaccines remains a main focus in the efforts to control malaria. Iron chelators have been studied as potential antimalarial drugs due to their activities against different stages of the parasite. The iron chelator FBS0701 affects the development ofPlasmodium falciparumearly gametocytes and lowers blood-stage parasitemia. Here, we tested the effect of FBS0701 on stage V gametocyte infectivity for mosquitoes. The incubation of stage V gametocytes for up to 3 days with increasing concentrations of FBS0701 resulted in a significant dose-related reduction in mosquito infectivity, as measured by the numbers of oocysts per mosquito. The reduction in mosquito infectivity was due to the inhibition of male and female gametocyte activation. The preincubation of FBS0701 with ferric chloride restored gametocyte infectivity, showing that the inhibitory effect of FBS0701 was quenched by iron. Deferoxamine, another iron chelator, also reduced gametocyte infectivity but to a lesser extent. Finally, the simultaneous administration of drug and gametocytes to mosquitoes without previous incubation did not significantly reduce the numbers of oocysts. These results show the importance of gametocyte iron metabolism as a potential target for new transmission-blocking strategies.

scholarly journals Mitosis in the Human Malaria Parasite Plasmodium falciparum

2011 ◽  
Vol 10 (4) ◽  
pp. 474-482 ◽  
Author(s):  
Noel Gerald ◽  
Babita Mahajan ◽  
Sanjai Kumar
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Complex Life Cycle ◽  
Anopheline Mosquito ◽  
Essential Requirement ◽  
Human Malaria Parasite ◽  
Content Type ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Key Events

ABSTRACT Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa ) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.

scholarly journals IgG Antibodies to Endothelial Protein C Receptor-Binding Cysteine-Rich Interdomain Region Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 Are Acquired Early in Life in Individuals Exposed to Malaria

2015 ◽  
Vol 83 (8) ◽  
pp. 3096-3103 ◽  
Author(s):  
Louise Turner ◽  
Thomas Lavstsen ◽  
Bruno P. Mmbando ◽  
Christian W. Wang ◽  
Pamela A. Magistrado ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Malaria Transmission ◽  
Severe Malaria ◽  
Erythrocyte Membrane ◽  
Protein C ◽  
Endothelial Protein C Receptor ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
Amino Terminal

Severe malaria syndromes are precipitated byPlasmodium falciparumparasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variantP. falciparumerythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of PfEMP1 proteins associated with severe malaria and found that the receptor for these PfEMP1 variants is endothelial protein C receptor (EPCR). The binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDR) of the subtypes α1.1 and α1.4 to α1.8. In this study, we investigated the acquisition of anti-CIDR antibodies using plasma samples collected in four study villages with different malaria transmission intensities in northeastern Tanzania during a period with a decline in malaria transmission. We show that individuals exposed to high levels of malaria transmission acquire antibodies to EPCR-binding CIDR domains early in life and that these antibodies are acquired more rapidly than antibodies to other CIDR domains. The rate by which antibodies to EPCR-binding CIDR domains are acquired in populations in areas where malaria is endemic is determined by the malaria transmission intensity, and on a population level, the antibodies are rapidly lost if transmission is interrupted. This indicates that sustained exposure is required to maintain the production of the antibodies.

scholarly journals Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

2013 ◽  
Vol 20 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Harold Obiakor ◽  
Marion Avril ◽  
Nicholas J. MacDonald ◽  
Prakash Srinivasan ◽  
Karine Reiter ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Chondroitin Sulfate ◽  
Erythrocyte Membrane ◽  
Recombinant Proteins ◽  
Binding Assay ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
First Time

ABSTRACTVAR2CSA, a member of thePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed inEscherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.

scholarly journals The Basal Complex Protein PfMORN1 Is Not Required for Asexual Replication of Plasmodium falciparum

mSphere ◽  
2021 ◽  
Author(s):  
Colleen J. Moran ◽  
Jeffrey D. Dvorin
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Severe Form ◽  
Blood Stage ◽  
Content Type ◽  
Human Malaria ◽  
Basal Complex ◽  
Complex Protein

Plasmodium falciparum parasites cause the most severe form of human malaria. During the clinically relevant blood stage of its life cycle, the parasites divide via schizogony.

scholarly journals Male and Female Plasmodium falciparum Mature Gametocytes Show Different Responses to Antimalarial Drugs

2013 ◽  
Vol 57 (7) ◽  
pp. 3268-3274 ◽  
Author(s):  
Michael J. Delves ◽  
Andrea Ruecker ◽  
Ursula Straschil ◽  
Jöel Lelièvre ◽  
Sara Marques ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Plasmodium Falciparum ◽  
Mature Male ◽  
Mammalian Host ◽  
Parasite Transmission ◽  
Significant Activity ◽  
Male And Female ◽  
Transmission Blocking ◽  
Content Type ◽  
Male Gametes

ABSTRACTIt is the mature gametocytes ofPlasmodiumthat are solely responsible for parasite transmission from the mammalian host to the mosquito. They are therefore a logical target for transmission-blocking antimalarial interventions, which aim to break the cycle of reinfection and reduce the prevalence of malaria cases. Gametocytes, however, are not a homogeneous cell population. They are sexually dimorphic, and both males and females are required for parasite transmission. Using two bioassays, we explored the effects of 20 antimalarials on the functional viability of both male and female mature gametocytes ofPlasmodium falciparum. We show that mature male gametocytes (as reported by their ability to produce male gametes, i.e., to exflagellate) are sensitive to antifolates, some endoperoxides, methylene blue, and thiostrepton, with submicromolar 50% inhibitory concentrations (IC50s), whereas female gametocytes (as reported by their ability to activate and form gametes expressing the marker Pfs25) are much less sensitive to antimalarial intervention, with only methylene blue and thiostrepton showing any significant activity. These findings show firstly that the antimalarial responses of male and female gametocytes differ and secondly that the mature male gametocyte should be considered a more vulnerable target than the female gametocyte for transmission-blocking drugs. Given the female-biased sex ratio ofPlasmodium falciparum(∼3 to 5 females:1 male), current gametocyte assays without a sex-specific readout are unlikely to identify male-targeted compounds and prioritize them for further development. Both assays reported here are being scaled up to at least medium throughput and will permit identification of key transmission-blocking molecules that have been overlooked by other screening campaigns.

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