A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasite's life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen-activated kinase-2 (Pfmap-2), a gametocyte-specific mitogen-activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap-2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.     

Targeting Human Transmission Biology for Malaria Elimination

Malaria remains one of the leading causes of death worldwide, despite decades of public health efforts. The recent commitment by many endemic countries to eliminate malaria marks a shift away from programs aimed at controlling disease burden towards one that emphasizes reducing transmission of the most virulent human malaria parasite, Plasmodium falciparum. Gametocytes, the only developmental stage of malaria parasites able to infect mosquitoes, have remained understudied, as they occur in low numbers, do not cause disease, and are difficult to detect in vivo by conventional methods. Here, we review the transmission biology of P. falciparum gametocytes, featuring important recent discoveries of genes affecting parasite commitment to gametocyte formation, microvesicles enabling parasites to communicate with each other, and the anatomical site where immature gametocytes develop. We propose potential parasite targets for future intervention and highlight remaining knowledge gaps.     

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.     

Malaria parasite growth is stimulated by mosquito probing

The ability of malaria parasites to respond positively to the presence of feeding mosquito vectors would clearly be advantageous to transmission. In this study, Anopheles stephensi mosquitoes probed mice infected with the rodent malaria parasite, Plasmodium chabaudi. Growth of asexual stages was accelerated and gametocytes appeared 1-2 days earlier than in controls. This first study, to our knowledge, of the effects of mosquitoes on 'in-host' growth and development of Plasmodium has profound implications for malaria epidemiology, suggesting that individuals exposed to high mosquito numbers can contribute disproportionately high numbers of parasites to the transmission pool.     

Impact of schistosome infection on Plasmodium falciparum Malariometric indices and immune correlates in school age children in Burma Valley, Zimbabwe

A group of children aged 6–17 years was recruited and followed up for 12 months to study the impact of schistosome infection on malaria parasite prevalence, density, distribution and anemia. Levels of cytokines, malaria specific antibodies in plasma and parasite growth inhibition capacities were assessed. Baseline results suggested an increased prevalence of malaria parasites in children co-infected with schistosomiasis (31%) compared to children infected with malaria only (25%) (p = 0.064). Moreover, children co-infected with schistosomes and malaria had higher sexual stage geometric mean malaria parasite density (189 gametocytes/µl) than children infected with malaria only (73/µl gametocytes) (p = 0.043). In addition, a larger percentage of co-infected children (57%) had gametocytes as observed by microscopy compared to the malaria only infected children (36%) (p = 0.06). There was no difference between the two groups in terms of the prevalence of anemia, which was approximately 64% in both groups (p = 0.9). Plasma from malaria-infected children exhibited higher malaria antibody activity compared to the controls (p = 0.001) but was not different between malaria and schistosome plus malaria infected groups (p = 0.44) and malaria parasite growth inhibition activity at baseline was higher in the malaria-only infected group of children than in the co-infected group though not reaching statistical significance (p = 0.5). Higher prevalence and higher mean gametocyte density in the peripheral blood may have implications in malaria transmission dynamics during co-infection with helminths.     

The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

ABSTRACT: BACKGROUND: Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. METHODS: The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. RESULTS: The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. CONCLUSIONS: The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Rodent malaria parasites Plasmodium chabaudi and P. vinckei do not increase their rates of gametocytogenesis in response to mosquito probing

Several vector-borne infectious agents facultatively alter their life history strategies in response to local vector densities. Some evidence suggests that malaria parasites invest more heavily in transmission stage production (gametocytogenesis) when vectors are present. Such a strategy could rapidly increase malaria transmission rates, particularly when adult mosquitoes begin to appear after dry seasons. However, in contrast to a recent experiment with a rodent malaria (Plasmodium chabaudi), we found no change in gametocytogenesis in either P. chabaudi or in another rodent malaria, P. vinckei, when their mouse hosts were exposed to mosquitoes. Positive results in the earlier study may have been because mosquito-feeding caused anaemia in hosts, a known promoter of gametocytogenesis. The substantial evidence that malaria and a variety of other parasites facultatively alter transmission strategies in response to a variety of environmental influences makes our results surprising.     

Adaptive changes in Plasmodium transmission strategies following chloroquine chemotherapy.

Both theory and data suggest that malaria parasites divert resources from within-host replication to the production of transmission stages (gametocytes) when conditions deteriorate. Increased investment into transmission stages should therefore follow subcurative treatment with antimalarial drugs, but relevant clinical studies necessarily lack adequate control groups. We therefore carried out controlled experiments to test this hypothesis, using a rodent malaria (Plasmodium chabaudi) model. Infections treated with a subcurative dose of the antimalarial chloroquine showed an earlier peak and a greater rate of gametocyte production relative to untreated controls. These alterations led to correlated changes in infectivity to mosquitoes, with the consequence that chloroquine treatment had no effect on the proportion of mosquitoes infected. Treatment of human malaria commonly does not result in complete parasite clearance. If surviving parasites produce compensatory increases in their rate of gametocyte production similar to those reported here, such treatment may have minimal effect on decreasing, and may actually increase, transmission. Importantly, if increased investment in transmission is a generalized stress response, the effect might be observed following a variety of antimalarial treatments, including other drugs and potential vaccines. Similar parasite life history counter-adaptations to intervention strategies are likely to occur in many disease-causing organisms.     

Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity

Malaria continues to devastate sub-Saharan Africa owing to the emergence of drug resistance to established antimalarials and to the lack of an efficacious vaccine. Plasmodium species have a unique streamlined purine pathway in which the dual specificity enzyme purine nucleoside phosphorylase (PNP) functions in both purine recycling and purine salvage. To evaluate the importance of PNP in an in vivo model of malaria, we disrupted PyPNP, the gene encoding PNP in the lethal Plasmodium yoelii YM strain. P. yoelii parasites lacking PNP were attenuated and cleared in mice. Although able to form gametocytes, PNP-deficient parasites did not form oocysts in mosquito midguts and were not transmitted from mosquitoes to mice. Mice given PNP-deficient parasites were immune to subsequent challenge to a lethal inoculum of P. yoelii YM and to challenge from P. yoelii 17XNL, another strain. These in vivo studies with PNP-deficient parasites support purine salvage as a target for antimalarials. They also suggest a strategy for the development of attenuated nontransmissible metabolic mutants as blood-stage malaria vaccine strains.     

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria 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.     

Human T clones reactive to the sexual stages of Plasmodium falciparum malaria. High frequency of gamete-reactive T cells in peripheral blood from nonexposed donors

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.