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Life, 49: 245 – 248, 2000 Copyright ° c 2000 IUBMB 1521-6543/00 $12.00 + .00 IUBMB

Hypothesis Paper Erythropoiesis and Molecular Mechanisms for Sexual Determination in Malaria Parasites R. E. L. Paul,1 C. Doerig, 2 and P. T. Brey1 1

Laboratoire de Biochimie et Biologie Mol´eculaire des Insectes, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France 2 INSERM U313, CHU Piti´e-Salpˆetri`ere, 91, Boulevard de l’Hˆopital, 75643 Paris Cedex 13, France

Summary Malaria parasites proliferate asexually within the vertebrate host but must undergo sexual reproduction for transmission to mosquitoes and hence infection of new hosts. The developmental pathways controlling gametocytogenesis are not known, but several protein kinases and other putative signal transduction elements possibly involved in this phenomenon have been found in Plasmodium. Recently, another developmental pathway, that of Plasmodium sex determination (male or female), has been shown to be triggered by erythropoiesis in the host. Rapid progress is being made in our understanding of the molecular basis of mammalian erythropoiesis, revealing kinase pathways that are essential to cellular responses triggered by the hormone erythropoietin. Although the molecular mechanisms whereby this hormone modulates the sex ratio of malaria parasites remain to be elucidated, it probably activates, within the parasite, transduction pathways similar to those found in other eukaryotes. Indeed, enzymes belonging to protein kinase families known to be involved in the response of mammalian cells to erythropoietin (such as the mitogen-activated protein kinases) have been identiŽ ed in P. falciparum gametocytes. Some of these enzymes differ markedly from their mammalian homologs; therefore, identiŽ cation of the transduction pathways of the parasite that are responsible for its developmental response to erythropoietin opens the way to the development of transmissionblocking drugs based on kinase inhibitors. IUBMB Life, 49: 245 – 248, 2000 Keywords

Erythropoiesis; Plasmodium; protein kinases; sex determination.

INTRODUCTION The spread of drug resistance in Plasmodium falciparum, the etiological agent of lethal human malaria, is currently one of the most serious public health issues facing the developing Received 22 February 2000; accepted 23 February 2000. Address correspondence to Dr. R. E. L. Paul. Fax: (33)-1-40-61-3471; E-mail: [email protected]

world. Vaccine and chemotherapeutic research has hitherto concentrated on the asexual parasite stages, which are responsible for its pathological effects. The failure of recent vaccines and the growing problem of drug resistance provided the impetus behind the Multilateral Initiative on Malaria in 1997, with the agenda to promote research towards the development of novel strategies for control. In particular, the Initiative called for investigations into “the complex relationship between malaria transmission rates, acquired immunity and the clinical forms, and incidence of the disease” (1). One direct consequence of this global push has been an increased research effort on the stages of the parasite involved in transmission. One of the goals of such investigations is the development of intervention strategies aimed at blocking the transmission of the parasite from humans to mosquitos and thereby interrupting the life cycle of the parasite (Box 1). SEXUAL DIFFERENTIATIO N IN PLASMODIUM Malaria parasites proliferate asexually within their vertebrate host, causing anaemia and disease. At some point during the infection, a portion of the parasites switch from asexual to sexual stage production. The sexual stages, gametocytes, are the sole forms of the parasite capable of infecting the mosquito vector and hence are essential for transmission and infection of new hosts. Until recently, gametocyte research has concentrated on the identiŽ cation of stage-speciŽ c gene expression and the molecular mechanisms underlying the developmental switches that result in the production of gametocytes from the asexual forms (reviewed in 2 – 4). Both ex vivo and in vivo studies point to environmental stimuli triggering the process of commitment to sexual stage production (gametocytogenesis) (5). Sexual commitment occurs most notably in response to environmental factors that either directly suppress asexual proliferation (e.g., antimalarial drugs) (6) or are associated with a worsening blood environment (e.g., fever responses and developing anaemia in clinical malarial episodes) (7). 245

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Box 1. Malaria Parasite Life Cycle ( 2, 4 ) The cycle starts when an infected female mosquito takes a bloodmeal. At blood-feeding, she injects saliva into the vertebrate host; the saliva contains an anaesthetic and an anticoagulant and, if infected with Plasmodium, will also unwittingly inject the parasite sporozoite stages, which invade vertebrate host cells (the liver in mammals and macrophages in birds). These sporozoites undergo asexual proliferation in the host cells, producing many hundreds of thousands of merozoite stage parasites about a week later. These merozoites invade erythrocytes and there they grow, divide asexually to produce further merozoites, and burst out from the cell to invade additional erythrocytes, a cycle occurring every 36 or 48 h, according to the Plasmodium species involved. These asexual blood stages are responsible for disease. At some point during the course of the infection, most notably, when such asexual proliferation is slowed, the merozoite stages grow but do not divide; at this point they produce the sexual stages, the gametocytes, which are gamete precursors. Transmission from the vertebrate host to the mosquito vector is mediated solely by these sexual stages of the parasite, which are distinguishable as males and females. Mature gametocytes are arrested in G0 of the cell cycle in the vertebrate host blood until they are taken up in the bloodmeal by another female mosquito, whereupon they transform into gametes: Each male gametocyte undergoes ex agellation, a process that produces as many as eight male gametes; each female gametocyte produces only one female gamete. Such gametogenesis occurs within 10 – 15 min after uptake in the mosquito bloodmeal—in response to the drop in temperature and the pH change associated with the change of hosts (from vertebrate to mosquito) and mosquito factors. Within 30 min the male must actively swim to Ž nd and fertilise the female gamete. The subsequent zygote transforms into a mobile ookinete that penetrates the mosquito stomach wall, where it encysts. Eight to 15 days later (depending on the Plasmodium spp.), this mature oocyst bursts, releasing several thousand sporozoites that invade the salivary glands of the mosquito and are injected into the vertebrate host during her next bloodmeal. Colonisation of a habitat by forms that proliferate asexually, combined with production of sexual stages in response to environmental degradation, is a strategy shared across the plant and animal kingdoms, whether in parasitic or free-living organisms (e.g., yeasts in suboptimal nutrient conditions [8], aphids in response to overcrowding, and the green alga Volvox in response to desiccation [9]). At the cellular level, responses to environmental changes are mediated by signaling pathways in which protein phosphorylation, in most cases, plays a crucial role. In Plasmodium, the instant environmental changes accompanying transmission from humans to mosquitos (change in temperature and

pH and exposure to xanthurenic acid, a mosquito-derived molecule [10]) stimulate the process of gametogenesis, also called gametocyte activation, whereby the gametocytes develop into male or female gametes (4). Similar phenomena occur in other protozoan blood parasites, the trypanosomatids (Trypanosoma and Leishmania spp.), where the change of host (human to insect vector) results in parasite stage differentiation accompanied by changes in protein phosphorylation (11). The expression of a panoply of protein kinases and other signal transduction elements in Plasmodium gametocytes suggests that intracellular signaling pathways similar to those regulating cellular differentiation in other eukaryotes may operate during Plasmodium sexual development (4, 12 – 14). ERYTHROPOIETIN AND SEX DETERMINATION IN PLASMODIUM In addition to commitment to sexual differentiation (gametocytogenesis) and gamete production (gametogenesis), a third developmental pathway operating in the sexual cycle of malaria parasites has recently been revealed: that which determines the sex of the gametocytes (male or female) (15). In vitro culture of P. falciparum has shown that the proportion of male and female gametocytes is Ž xed, clone-speciŽ c, and therefore genetically determined (16). However, in vivo, sex determination is under secondary control, changing in response to the haematological state of the individual, speciŽ cally, the host’s erythropoietic state (15). During the course of a malaria infection in the vertebrate host, the blood environment becomes increasingly deleterious for the parasite: Accompanying the increasing anaemia (caused by parasite-induced haemolysis) is a developing immune response against the parasite. Until recently, the effect of the host’s responses on parasite sexual development was thought to be restricted to stimulation of gametocytogenesis (2 – 7). However, it has now been established that the infected host’s anaemia triggers erythropoiesis and that the ensuing increase in erythropoeitin (Epo)3 concentrations in blood results in a shift in the sex ratio of gametocytes in favor of males (microgametocytes ). That there should be more male than female mature gametocytes is important to the parasite because the immune response, although apparently ineffective against the gametocytes within the vertebrate blood system, reduces Plasmodium fertilisation efŽ ciency (17). Fertilisation occurs after gametogenesis within the bloodmeal of the engorged mosquito, where a male gamete actively swims to Ž nd and fertilise a female (Box 1). Antibodies in the ingested blood agglutinate the male gametes (18) and therefore speciŽ cally affect the capacity of the males to Ž nd females. As if compensating for this reduced male gamete efŽ ciency, the parasite increases its male gametocyte production within the vertebrate host by using Epo as a signal. Whether this developmental switch for sex determination occurs simultaneously 3

Abbreviations: Epo, erythropoietin; Epo-R, erythropoietin receptor; IL, interleukin; MAPK, mitogen-activated protein kinase.

SEXUAL DETERMINATION IN PLASMODIUM

with the switch to sexual commitment seems increasingly probable, because all sexually committed asexual stages (merozoites) produced from a single infected erythrocyte (hence the schizont parasite stage) become either male or female (19). However, the precise timing of commitment remains unknown, although it probably occurs in the asexual parasite stages (trophozoites ) before merozoite release (20, 21). HOW DOES EPO MEDIATE PLASMODIUM SEX DETERMINATION? Epo is the major glycoprotein hormone triggering erythropoiesis in mammals (22). The action of Epo on responsive mammalian cells is initiated by the binding of the hormone to the Epo receptor (Epo-R; a member of a subfamily of cytokine receptors: interleuken [IL]-2-R, IL-3-R, IL-4-R, and Epo-R), which results in a multiple-step transduction cascade that activates well-deŽ ned protein kinases, such as mitogen-activated protein kinases (MAPKs) and Jun-ATF kinases (23 – 25). Substrates for these kinases include transcription factors that, when activated, are responsible for the expression of genes associated with the adaptative response to the hormone. Whether Plasmodium expresses an endogenous Epo-R is currently unknown, and available data do not establish whether the parasite responds directly to the hormone or not (the absence of Epo-R in reticulocytes and erythrocytes [26] may facilitate the search for a parasite-derived receptor). In contrast, two MAPK homologs of the ERK1/ERK2 subfamily have been found to be expressed in Plasmodium blood stages: Pfmap-1, which is found in both asexual parasites and gametocytes (14, 27), and Pfmap-2, which appears to be expressed speciŽ cally in gametocytes (28). The function of these enzymes in parasite development is not known; an attractive hypothesis is that one of them may transmit the sex determination signal from Epo to the nucleus. The tools to validate this hypothesis are now available, because the activation of recombinant plasmodial MAPKs by upstream kinase activities present in parasite extracts can be measured, as has been shown for Pfmap-2 (28). This approach can in principle be followed for any candidate kinase. Once a kinase that responds to Epo has been identiŽ ed, intermediate elements that lie in the pathway between the input (hormone treatment) and the output (kinase activation) can be investigated in several ways, for example, by determining whether inhibitors of a class of enzymes thought to be involved abolish the effect of Epo on the kinase. Targeted gene disruption, which is well suited for studying genes that are essential for sexual development but dispensable for asexual multiplication, is another approach now available to determine whether these enzymes are required for sex determination. Although transgenic Plasmodium technology is still in its infancy (29), this approach, which has already revealed the importance of kinases in Leishmania (30), should enable assessment of the role of kinase pathways in Plasmodium sexual differentiation. Targeted gene disruption in both mouse and zebraŽ sh models has revealed several genes essential for erythropoiesis (26), and our comprehension of the essential genes and regulatory networks involved in erythropoiesis is advancing rapidly (23 – 26, 31). Recent adaptation

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of differential display (32) and microarray technologies (33) for malaria research, coupled with the P. falciparum genome project (www.wehi.edu.au/MalDB-www/who.html), now puts the identiŽ cation and characterisation of the stage-speciŽ city of malaria gene expression within our grasp. POSSIBLE APPLICATIONS TO MALARIA CONTROL Structural and functional differences between plasmodial kinases and their mammalian homologs offer an opportunity for selective inhibition (34). Indeed, the homologs of a speciŽ c kinase, cyclin dependent kinase 1 (CDK1) from phylogenetically distant organisms have been shown to differ considerably in their susceptibilities to a given inhibitor (35). The plasmodial MAPKs share only » 45% identity with mammalian MAPKs and differ in other important aspects (e.g., the activation site of Pfmap-2) that suggest their modes of regulation differ signiŽ cantly one another. The fact that recombinant plasmodial MAPKs (and other kinases) display kinase activity in vitro opens the way to identifying inhibitors from chemical libraries, the most potent of which can then be assayed for effect on sexual development of the parasite. A similar approach can in principle be taken for any elements (not necessarily protein kinases) required for Epo-mediated sex determination. IdentiŽ cation of Epo-R analogs and associated pathways in P. falciparum will be considerably facilitated at completion of the genome project. The elucidation of these pathways has already been simpliŽ ed by the development of now routine in vitro culture techniques with the capability of stagespeciŽ c puriŽ cation (36). The principle of developing antimalarial drugs targeting speciŽ c parasite developmental pathways is a step forward from the current state-of-the-art drugs, which are predominantly variations on the theme of naturally occurring compounds, with no or only rudimentary comprehension of their mode of action. P. falciparum has shown a remarkable ability to develop resistance to antimalarial drugs. However, drug resistance arises by selection under drug pressure of naturally occurring mutants (37). Because of the nondividing nature of gametocytes, such selection and ampliŽ cation of resistant parasites will presumably be much slower with respect to drugs that target sexual stages. Knowledge of the molecular mechanisms of sex determination offers an exciting possibility: that of blocking transmission by “tricking” the parasite into producing gametocytes of only one sex. Such manipulation of the parasite’s natural transmission pathways should make countermeasures by the parasite less effective and thus prolong the usefulness of such potential control methods. REFERENCES 1. Anonymous . (1997) Briefmalaria: Time to put malaria control on the global agenda. Nature 386, 535 – 540. 2. Carter, R., and Graves, P. M. (1988) Gametocytes. In Malaria: Principles and Practice of Malariology, Vol. 1 (Wernsdorfer, W. H., and McGregor, I. A., eds.). pp. 253 – 306, Churchill Livingstone, London. 3. Alano, P., and Carter, R. (1990) Sexual differentiation in malaria parasites. Annu. Rev. Microbiol. 44, 429 – 449.

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