Molecular & Biochemical Parasitology 172 (2010) 57–65

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Molecular & Biochemical Parasitology

Review Malaria gametocytogenesis

David A. Baker ∗

Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK article info abstract

Article history: Male and female gametocytes are the components of the malaria parasite life cycle which are taken Received 22 January 2010 up from an infected host bloodstream by mosquitoes and thus mediate disease transmission. These Received in revised form 29 March 2010 gamete precursors are morphologically and functionally quite distinct from their asexual blood stage Accepted 30 March 2010 counterparts and this is reflected in their distinct patterns of gene expression, cellular development and Available online 8 April 2010 metabolism. Recent transcriptome, proteome and reverse genetic studies have added valuable informa- tion to that obtained from traditional studies. However, we still have no answer to the fundamental Keywords: question regarding sexual development: ‘what triggers gametocytogenesis’? In the current climate of Malaria Plasmodium eradication/elimination, tackling transmission by killing gametocytes has an important place on the Gametocyte agenda because most antimalarial drugs, whilst killing asexual blood stage parasites, have no effect on Parasite the transmissible stages. Drug © 2010 Elsevier B.V. Open access under CC BY license. Differentiation

Contents

1. Introduction ...... 57 2. The distinct morphology of gametocytes compared to the asexual blood stages is reflected in their unique pattern of gene expression...... 58 3. Gametocyte metabolism ...... 58 3.1. Nucleic acid synthesis ...... 58 3.2. Carbohydrate metabolism and energy production ...... 59 3.3. The mitochondrion and apicoplast ...... 59 4. What causes gametocytogenesis? ...... 59 5. P. falciparum isolates vary in their capacity to produce gametocytes in the laboratory ...... 60 6. Sex ratios ...... 61 7. Gametocyte proteins with a subsequent role in fertilisation ...... 61 8. Cytoadherence and sequestration ...... 62 9. Drugs that affect gametocyte development and reduce transmission ...... 62 10. Concluding remarks ...... 63 Acknowledgements ...... 63 References ...... 63

1. Introduction parasites within the red blood cells of a human host leads to the pathology and symptoms of malaria. The malaria parasite life cycle Malaria is caused by protozoa of the genus Plasmodium; these also comprises a phase of sexual reproduction which takes place are apicomplexan parasites and are part of the alveolate group- in the mosquito vector, but the actual switch to sexual develop- ing [1]. As well as other animal pathogens such as Toxoplasma, ment occurs in the vertebrate host, with the formation of male Crytptosporidium, Theileria and Eimeria, alveolates also comprise and female gamete precursors (gametocytes), and is a prerequi- free living ciliate protozoa such as Paramecium and Tetrahy- site for transmission of disease [2]. This review will focus on our mena and also the dinoflagellates. Asexual proliferation of malaria current knowledge of the biology of gametocytes and their devel- opment, and will highlight how the parasite genome sequence and post-genomic approaches have advanced our understanding ∗ Tel.: +44 (0)20 7927 2664; fax: +44 (0)20 7636 8739. of this important life cycle stage. The majority of the information E-mail address: [email protected]. has been derived from research carried out on the major human

0166-6851© 2010 Elsevier B.V. Open access under CC BY license. doi:10.1016/j.molbiopara.2010.03.019 58 D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 pathogen Plasmodium falciparum where the blood stages can read- which determine the characteristic shape of gametocytes. The ily be cultured in vitro [3], but also the rodent malaria parasite highly distinct morphology and, to some extent, the extended Plasmodium berghei which is a genetically more amenable system period of development made it obvious that P. falciparum game- and provides an important in vivo model [4]. It is encouraging that tocytes would express a large number of proteins specific to this advances in our knowledge are being made continuously, though stage of development. It also became clear that sexual stage spe- consequently a review can provide a snapshot at best. The nature cific promoters were present that regulate the precise temporal of reverse genetic techniques currently applicable to Plasmodium expression of genes. For example, in one of the first studies to exam- provided a window of opportunity to advance the study of sexual ine this issue, transfection of Plasmodium gallinaceum with reporter stage genes and has facilitated recent progress. The recent funding constructs containing a segment of 5 UTR of the Pfs16 and Pfs25 injection to support malaria elimination/eradication efforts and the genes upstream of GFP was sufficient to direct expression in a sex- resulting growing recognition of the importance of tackling trans- ual stage specific manner, and also governed appropriate temporal mission [5,6] will provide an opportunity to further increase our expression [19]. understanding of the biology of these fascinating life cycle stages. Examination of the P. falciparum proteome detected more than 900 proteins in gametocytes; 315 of these were found exclusively in gametocytes. More than 600 proteins were found in gametes 2. The distinct morphology of gametocytes compared to and 97 of these were gamete-specific [20]. Around 250–300 genes the asexual blood stages is reflected in their unique pattern specifically upregulated at the mRNA level during P. falciparum of gene expression gametocyte development have been detected by transcriptome analysis [11,21]. A sex-specific proteome study in P. berghei has Though a large proportion of the work on Plasmodium gameto- shown that a large proportion of the 5–600 proteins are expressed cytes has been carried out on P. falciparum, the extended period exclusively in either male or female gametocytes and relatively (8–12 days) of development (see [6] for review) is unique to this few are expressed in both, reflecting the individual roles of the species (and the closely related primate malaria parasite Plasmod- two sexes. This study utilised the fact that gametocytes have sex- ium reichenowi [2]). Therefore some of the findings may not be specific promoters and two of these were used to drive expression relevant to other malaria species. P. falciparum gametocyte devel- of distinct fluorescent tags allowing cell sorting [22]. Proteins iden- opment can be conveniently divided into five morphologically tified in males included those that will subsequently be involved recognisable stages (I–V) according to a widely used classifica- in axoneme formation and motility, but also DNA replication that tion system [7]. This system documents the distinctive changes in follows activation. The distribution of mitochondrial enzymes and appearance that occur following invasion of a red cell by a sex- proteins involved in protein synthesis were biased towards female ually committed merozoite. After the (sexually committed) ring gamete preparations. Sexual dimorphism is morphologically most stage, the gametocyte grows and elongates to gradually occupy the apparent from stage IV onwards in P. falciparum and this stage majority of the host red cell. Stage I gametocytes (up to ∼40 h post- is characterised by an elongated shape with both ends pointed. invasion) are difficult to distinguish from young trophozoites in a The female gametocyte or ‘macrogametocyte’ is characterised by Giemsa-stained blood film, but are roundish and a pointed end and a relatively small nucleus (with a nucleolus) and concentrated pig- distinctive pigment pattern may be visible. Specific antibodies to ment pattern; by contrast the nucleus is larger (apparently lacking a early gametocyte markers using IFA are required to be 100% certain nucleolus) and the pigment more diffuse in the male (‘microgame- of the identity of stage I gametocytes. For many years the membrane tocyte’). The frequency of electron dense osmiophilic bodies at the protein Pfs16 has been a valuable marker of gametocytogenesis; its periphery of the cytoplasm is significantly higher in female game- expression has been measured at 24 h post-invasion [8] and per- tocytes. Secretions from osmiophilic bodies are involved in host sists throughout gametocyte development. Electron microscopy membrane disruption during gamete emergence from red blood revealed that this protein is located in the parasitophorous vacuole cells [23,24] and deletion of Pfg377 has revealed a role for this pro- membrane (pvm) as well as in the membrane surrounding ingested tein in osmiophilic body formation and female gamete emergence food vacuoles and in membranous clefts in the red cell cytoplasm. [25]. Ribosomes are also found at higher densities in female game- The protein is lost from the parasite during gametocyte activation tocytes accounting for their bluer appearance in Giemsa-stained as the pvm is disrupted in characteristic multilaminate whorls and blood films (pink in males). Stage V or mature gametocytes have makes way for the emerging gametes [9,10]. It has been noted that rounded ends due to depolymerisation of the sub-pellicular micro- other early gametocyte proteins Pfpeg3 and Pfpeg 4 expressed in tubules [18] and may be sausage or crescent-shaped. stage II gametocytes [11]; PF14 744 and PF14 748 [12] are also located in the pvm. The common pvm location of all these early proteins has been suggested to reflect the requirement to remodel 3. Gametocyte metabolism this host-derived capsule in a manner distinct from that required in asexual blood stage parasites [13]. Another early gametocyte spe- 3.1. Nucleic acid synthesis cific protein (Pfg27) is transcribed at ∼30 h post-invasion [14]. This is a cytoplasmic RNA-binding phosphoprotein [15] that intriguingly There are intriguing earlier observations suggesting the possi- has no orthologue in other malaria parasite species (apart from P. bility of DNA synthesis and nuclear division in P. falciparum and P. reichenowi), leading to the hypothesis that it may have a role in the berghei gametocytes [18,26]. However, current opinion is that there extended period of gametocyte development. Deletion of this gene is no replication of the genome in gametocytes; tritiated hypoxan- allows only a fraction of gametocytes to develop normally, but most thine is apparently not taken up by gametocytes [27]. It is likely Pfg27− gametocytes are morphologically abnormal [16]. that nucleic acid synthetic activity in gametocytes is restricted to Gametocyte stages II–V can all be readily distinguished in RNA synthesis and that, consistent with genetic evidence, gameto- Giemsa-stained blood films with practise and their morpholo- cytes are haploid and that rapid genome replication and nuclear gies have been depicted elsewhere [13,17]. The individual stages division occur in male gametocytes only after activation in the of gametocyte development have also been described at the mosquito midgut. In the vertebrate host, gametocytes appear to ultrastructural level by Sinden [18]. This EM-based study gives be developmentally arrested at the G0 phase of the cell cycle. details of the development of the sub-pellicular microtubule-based Synthesis of RNA is thought to occur up to around day 6 of cytoskeleton and the surrounding interrupted, double membrane P. falciparum gametocyte development and this (as well as pro- D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 59 tein synthesis and haemoglobin digestion) is reported to cease the mitochondrion and apicoplast have revealed that in both cases, in mature gametocytes [28,29]. Experimentation with northern there is only one of each of these organelles in both male and female blots and gametocyte cDNA libraries in the late 1980s- early 1990s gametocytes. The apicoplast does not show significant morpholog- revealed a surprisingly high proportion of transcripts to be ‘sex- ical changes during gametocytogenesis and remains small which ual stage specific’. It was puzzling for example that so many genes contrasts remarkably with the development of the mitochondrion. encoding putative signalling enzymes (e.g. protein kinases) should This consists of a large branching structure observed from stage II be expressed in this so-called quiescent stage of the life cycle. onwards which appeared to envelop the apicoplast with which it The explanation began to emerge-that some mRNAs such as those remains closely associated and this likely facilitates (their known) encoding p25 and p28 [30–32] were synthesised in female game- metabolic cooperation. This branched structure probably accounts tocytes, but translation was repressed until subsequent mosquito for previous reports of parasites having multiple mitochondria stages of the life cycle. It also became clear that this phenomenon in electron micrographs. This study also demonstrated that both was likely to apply to numerous transcripts in both P. falciparum and the apicoplast and the mitochondrion remain in the remnants of P. berghei gametocytes [33,34]. Details of the translational repres- the activated microgametocyte confirming that the male gamete sion mechanism were later revealed. Immunoprecipitation was lacks both and supports the evidence that these organelles are used to show association of P. berghei p25 and p28 mRNA (and maternally inherited [43,44]. It has been reported that gametocyte others) in complex with DOZI that is encoded by an orthologue mitochondria have tubular cristae [45], a key diagnostic feature of enzymes in the DDX6 family of DEAD-box RNA helicases. Their of alveolates, which may have implications for function. Others roles in other organisms include storage of key mRNAs in transla- have stated that the mitochondria of mammalian Plasmodium are tionally silent ribonucleotide particles (mRNPs) and translational initially acristate, but develop cristae later at the ookinete stage repression [35]. DOZI was highly upregulated in the proteome [46]. analysis of female gametocytes [22]. Deletion of the pbdozi gene It is widely viewed that in asexual blood stage parasites, mito- prevented ookinete formation; development of zygotes arrested chondria are unable to carry out full oxidation of glucose for development prior to meiosis [35]. In this mutant line, a total of mitochondrial ATP synthesis and it has been suggested that the 370 transcripts were shown to be down regulated significantly in main function of the mitochondrial TCA cycle is production of gametocytes (interestingly 92 transcripts increased in abundance) succinyl Co-A for haem biosynthesis. It was also pointed out compared to wild type parasites using microarray analysis, includ- that an important TCA entry point could be via glutamate from ing the majority of those transcripts previously hypothesised to haemoglobin degradation [47]. It is also clear that a key role of undergo translational repression [33]. It is likely that translational the mitochondrial electron transport chain is production of orotate repression allows stock piling in the gametocyte of mRNAs that can to fuel cytoplasmic pyrimidine biosynthesis [47,48]. However, it is be rapidly translated once the parasite receives the environmental thought that other dehydrogenases in the mitochondria are likely cues required to trigger gametogenesis. to play additional essential metabolic roles in the malaria para- A unique feature of malaria parasites is the developmentally site [49]. In a study examining transcripts present at the various regulated expression of the dispersed, nuclear encoded small sub- stages of gametocyte development 15 of the 16 mRNAs encod- unit ribosomal RNA (SSU rRNA) genes [36]. There are seven of ing enzymes of the mitochondrial TCA cycle were upregulated in these in P. falciparum and four in the rodent malarias. Only A-type gametocytes [21] and nine of these were detectable at the protein rRNA is expressed in asexual blood stage parasites. This form is level in female P. berghei gametocytes and five in male gameto- also expressed in gametocytes but also precursors of the S-type cytes in a sex-specific proteome study [22]. The latter sex-specific rRNA (formerly known as the C-type) are expressed in gametocytes proportion may have functional significance. Expression of these and are processed in subsequent mosquito stages and replace the genes and potential enzyme activity is consistent with the pres- dominant A-form rRNA population at around 6 days after fertilisa- ence of a spectacularly expanding mitochondrion in developing P. tion corresponding with development of sporozoites. It seems that falciparum gametocytes [42]. Clearly there is a requirement for a the switch between ribosome populations coincides with the dis- careful biochemical/genetic examination of mitochondrial function tinct environmental transitions that the parasite encounters as it in gametocytes and mosquito stages. It will also be important to progresses through the life cycle. determine whether functionality varies between malaria parasite species. 3.2. Carbohydrate metabolism and energy production There is evidence that intermediates in haem biosynthesis are transferred between the mitochondrion and the apicoplast [50]. It has long been known that the malaria parasite relies on Interestingly mRNAs corresponding to three of the six genes impli- glucose for energy production via anaerobic glycolysis and that cated in the haem biosynthesis pathway were upregulated during parasite derived enzymes are involved [37–40]. More recent identi- gametocytogenesis as well as six members of the type II fatty acid fication of all the corresponding genes in the parasite genomes has biosynthesis pathway [21]. In the P. berghei sex-specific proteome supported the biochemical evidence [41]. Organic products (such study [22] it is interesting to note that apicoplast proteins are as lactate) are generated rather than complete degradation to CO2 expressed at equivalent levels in males and females indicating that and H2O. A trend towards decreased expression of genes involved these proteins are likely to be active in the male gametocyte itself in glycolysis, protein biosynthesis and haemoglobin catabolism rather than solely in readiness for later development (since the was observed in later gametocyte stages [21] likely reflecting a apicoplast is absent in the male gamete). decrease in certain metabolic processes with increasing age and corresponding to the absence or reduced gametocytocidal activity of antimalarial drugs targeting some of these processes (see later 4. What causes gametocytogenesis? section). It is likely that progression through the complex series of stages 3.3. The mitochondrion and apicoplast of the malaria parasite life cycle involves an intricate interplay between the parasite’s changing environment and its own in-built A recent study has revealed important information on the mor- genetic programme. Some of the life cycle transitions coincide with phology of the mitochondrion (and apicoplast) in gametocytes [42]. extreme changes in environment that are easy to envisage causing Using a GFP tagging approach and specific antibodies, markers of dramatic changes in patterns of gene expression. Examples include 60 D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 when a gametocyte is transported from the bloodstream into the based on the evidence that in at least some P. falciparum infections, mosquito midgut, or the sporozoite as it leaves the mosquito sali- gametocytogenesis can be triggered at the onset of infection prior vary glands and enters the mammalian bloodstream. Changes in to the onset of symptoms [75].Sofarin vivo studies in humans have temperature, pH, salt composition and the presence of e.g. organic not helped to solve the problem of what triggers gametocytogen- chemical signals will be sensed, transduced and patterns of bio- esis because it is not possible to predict when or if gametocytes chemical activity changed rapidly. However, one dramatic life cycle will develop during the course of a P. falciparum infection; this transition that does not require an abrupt change of environment varies between individual patients and many host factors could (due to the absence of a change in location or host) is the ‘decision’ potentially influence the rate of conversion of asexual parasites to trigger the genesis of gametocytes. to gametocytes [17]. The study of gametocytogenesis in vivo is Numerous papers have described manipulation of culture con- also complicated by the fact that the developing stages sequester ditions and addition of pharmacological agents which can increase and only mature forms are present in the peripheral circulation. gametocyte numbers or apparently initiate gametocytogenesis. For The stage of an infection at which gametocytes first appear, con- example, addition of red cell lysate has been shown to increase trasts between P. falciparum and Plasmodium vivax. Gametocytes the rate of gametocytogenesis [51] as has the presence of human can appear simultaneously with asexual parasitaemia in P. vivax serum and lymphocytes [52], mammalian hormones [53,54], high perhaps indicating that liver stage schizonts as their source. In levels of reticulocytes [55], some inhibitors of nucleic acid syn- P. falciparum infections, the appearance of gametocytes is usually thesis [56] including antifolates [57] and also chloroquine in later and more sustained. However, the relatively late appearance [58–60]. Furthermore it has also been reported that apparent is expected since the maturation period of gametocytes is much gametocyte non-producers can be stimulated to produce game- longer for P. falciparum. tocytes by addition of Berenil and ammonia compounds [56,61]. Discovery of the molecular mechanism that triggers gametocy- It has been suggested that haematological disruption, i.e. red cell togenesis is arguably the ‘Holy Grail’ of malaria parasite biology. lysis, anaemia, dyserythropoiesis and reticulocytosis is the key to Once this is achieved it may simultaneously give insight into the changes that are linked to gametocyte production [62]. There is secrets of sex determination in the parasites. When pulse field gel evidence that the appearance of gametocytes in P. falciparum cul- electrophoresis became available to physically separate chromo- tures correlates with high asexual parasite density [63]. It has also somes, the possibility of sex chromosomes was explored, but it been shown that diffusible factors in cultures can favour game- became clear that these do not exist in malaria parasites and that tocytogenesis [64]. However, it has further been suggested that gametocyte-specific and sex-specific genes are dispersed amongst diffusible factors from asexual blood stage parasites in log phase the 14 chromosomes. It has also been clear for many years that all may repress gametocytogenesis, but when parasitaemia reaches the necessary information to form both female and male gameto- a critical threshold, this repression is lifted. It was postulated cytes is carried within a single cloned asexual blood stage parasite. that gametocytogenesis might actually be the default developmen- In P. falciparum the decision to become a gametocyte appears to tal pathway which is reminiscent of the fact that all merozoites be made in the asexual cycle prior to the one where gametocytes of haemoproteids (apicomplexan relatives), following invasion of are formed. Current in vitro evidence supports the view that a avian red blood cells, become gametocytes by default [6].Inmem- P. falciparum merozoite is pre-committed to sexual development bers of the more distant alveolate ciliate relatives, helical protein before egress from the schizont. As a result, it appears that all ‘pheromones’ are secreted that interact with other cells to cause the merozoites derived from a single schizont, will become either growth arrest and they become sexual forms [65]. gametocytes or asexual trophozoites [63] and furthermore evi- There are also reports of potential signalling pathways that may dence suggests that all the merozoites from a sexually committed be involved in triggering gametocytogenesis. For example, phor- schizont will become either male or female [76,77]. It has been bol ester inducing pathways [66] and the cAMP signalling pathway shown using plasmids expressing GFP under the control of sexual [66–70]. An intriguing increase in conversion to sexual develop- stage specific promoters (PF14 0748 [12]; Pfs16 (C. Swales and D. ment was observed on addition of cholera toxin to P. falciparum Baker, unpublished data); Pfg27 (A. Olivieri and P. Alano, unpub- cultures [71]. The conclusion was that a G protein-dependent sig- lished data)) that these promoters are active in a sub-population of nalling system may mediate the switch to sexual development in segmented schizonts the Pfs16 promoter. This supports the view response to environmental factors. The apparent identification of that the sexual switch has taken place prior to merozoite release in heterotrimeric G␣ subunits in this study is puzzling due to the the asexual cycle preceding gametocyte formation. lack of obvious encoding genes in the Plasmodium genome data. Whether the later report of involvement of host red blood cell G protein signalling in P. falciparum infection can reconcile these 5. P. falciparum isolates vary in their capacity to produce observations remains to be seen [72]. Interestingly, the Plasmodium gametocytes in the laboratory genomes encode potential homologues of G protein-coupled recep- tors from the plant, Arabidopsis thaliana, that have been shown to It is clear that some laboratory adapted isolates can readily have both intrinsic GDP/GTP exchange and GTPase activities; thus produce gametocytes in vitro and others cannot. Clone 3D7 and potentially functioning as ‘hybrid’ G protein-coupled receptors/G␣ its parental isolate NF54 are very reliable and widely used game- subunits [73] (Joanne Thompson, personal communication). tocyte producer lines. However, they lose the ability to produce However, none of these studies have given conclusive insight gametocytes with increasing in vitro passage number [78] suggest- into the physiological mechanism by which the parasite switches ing that parasites that undergo only asexual division are selected to sexual development. It is possible that there may not be a single and that these might have lost genes necessary for sexual develop- mechanism and that multiple factors may contribute to the deci- ment. It was reported that P. falciparum isolates with a short form of sion to form gametocytes. A thought provoking model for switching chromosome 9 (using Southern analysis of pulsed field gel separa- to sexual development has been proposed which requires that indi- tions) had lost [79] (or greatly reduced [80]) the ability to produce vidual parasites vary in their sensitivity to the environment [74]. gametocytes (and concomitantly cytoadherence) suggesting that Sexual commitment is also viewed by some as a stress response that a gene or genes in the absent segment of the chromosome might allows the parasite to escape from an increasingly unfavourable be involved in gametocytogenesis (and cytoadherence). Interest- environment. However, an alternative view is that the stress asso- ingly, selection of adherent parasites on myeloma cells (expressing ciated with gametocyte production may not be causal. This view is CD36), also restored the ability to produce gametocytes (by a fac- D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 61 tor of 10) and these parasites had a full length chromosome 9 [81]. are regulated has been the subject of much research and debate. Subsequent work analysed genes in this region of chromosome 9 Sex ratios are thought to vary in response to environmental fac- including Pfgig [82]. Transformants in which Pfgig had been dis- tors that influence fertility and also in response to the number rupted showed a reduced ability (∼5-fold) to produce gametocytes. of genotypes in an infection and that this ratio affects transmis- Whether this sheds light on the mechanism of gametocytogenesis sion success [90]. Conditions which adversely affect male fertility or blockage of one step during gametocyte development is not yet redress the balance favouring male production. Induction of ery- known. The gradual loss of the ability of P. falciparum to produce thropoesis in the host has been shown to increase the proportion gametocytes in culture over time makes it very difficult to identify of male gametocytes produced in P. gallinaceum suggesting that genes that are involved in gametocytogenesis using a gene knock- host hormones can influence sex ratios [91]. Recent experimental out approach due to the extended period of drug selection required. data have indicated that rodent malaria parasites, like multicel- Therefore complementation experiments that reverse the ‘inability lular organisms, ‘obey the rules’ of the sex allocation theory and to produce gametocyte phenotype’ by reintroduction of the gene sex ratio is an important determinant of fitness in P. chabaudi. Par- into the actual knockout line are required. Recently it has been asites appear to be able to detect the presence of both identical shown that complementation of a gametocyte-deficient parasite and co-infecting genotypes and to vary their sex ratio accordingly line that has a 19 kb deletion in this region of chromosome 9 with during the course of infection; in multiple infections the sex ratio an open reading frame, designated Pfgyi1 and previously known tends towards 1:1 [92]. It will be extremely interesting to deter- as cytoadherence-linked protein normally found in this segment, mine which parasite molecules monitor environmental conditions rescues the phenotype [83]. It is therefore clear that expression and recognition of non-identical phenotypes in order to influence of this gene is required for gametocyte production. The ability to sex determination. retain gametocyte production in P. falciparum lines by selection of cytoadherent parasites (to reduce loss of chromosome ends) on e.g. Gelofusin et al. [84] is both an interesting observation in this context 7. Gametocyte proteins with a subsequent role in and a very useful tool. It is also clear that laboratory adapted lines fertilisation exist in which gametocytogenesis is blocked at different stages. For example, clone K1 and clone T996 have been described as ‘game- The peripheral membranes of a gametocyte comprise the outer- tocyte non-producers’. Examination of these clones by IFA with most red blood cell membrane, the pvm and the plasma membrane. a monoclonal antibody to Pfs16 (an early marker of gametocyto- The gametocyte also has a pair of incomplete membranes beneath genesis) shows that stage I gametocytes are quite abundant, but the plasma membrane surrounding the pellicle. This double mem- are not apparent by Giemsa staining (unpublished data). In these brane is absent from all asexual blood stage parasites apart from lines, the ‘block’ has occurred sometime after the initial switch to merozoites. A number of proteins have been characterised that sexual development. Other truly gametocyte non-producer lines are located on these various membranes. Pfs48/45 and Pfs230 have been produced in P. falciparum [23] and P. berghei [85] are located as a complex on the plasma membrane of the game- and have proved very useful tools for investigating gametocyte tocyte (see [93] for review) with only Pfs48/45 being directly biology. anchored to the membrane by a GPI moiety. Following activation It is possible that the inability to produce gametocytes in vitro of gametocytes in the mosquito midgut, these proteins become may not be due to deletion of one or more genes, but to muta- directly exposed to the blood meal on the extracellular male and tion of key genes or promoter regions that influence the actual female gamete. It is thought they play a crucial role in fertili- switch to sexual differentiation or its progression. Alternatively, sation. Monoclonal antibodies raised to these proteins are able an epigenetic mechanism might be involved in regulation of game- to block transmission when fed to mosquitoes using an artificial tocytogenesis that is similar to that demonstrated for the mutually membrane feeding apparatus and they have long been viewed as exclusive expression of subtelomeric var genes [86,87]. Here inac- promising transmission blocking vaccine candidates (see [94] for tive var genes are located at the nuclear periphery and silenced review). Whilst there is a high degree of conservation of the protein by association with PfSir2. Derepression of transcription occurs sequences between diverse isolates, there are a small number of following exit from the PfSir2 cluster. It is possible that if genes substitutions in geographically distinct populations [95]. Deletion involved in the switch to gametocytogenesis are located in sub- of the gene encoding P48/45 in both P. berghei and P. falciparum has telomeric regions, then they may be regulated in a similar way. demonstrated that the protein is an important determinant of male A defect in this epigenetic regulatory mechanism might explain fertility [96]. Deletion of Pfs230 has revealed a role for this protein why isolates lacking deletions cannot produce gametocytes (Cathy in the characteristic agglutination of extracellular gametes with Taylor, PhD thesis, 2007). A recent study using a histone deacety- uninfected red blood cells observed during exflagellation. Pfs230− lase inhibitor in combination with global transcriptional analysis mutant males develop normally, but are unable to bind to red strongly suggests that epigenetics plays an important role in stage cells within centres of exflagellation following gametocyte activa- specific gene expression in P. falciparum [88]. tion, resulting in a significant reduction in mosquito infection after membrane feeding [97]. There are also a group of six adhesive pro- teins (PfCCP family [98] or Lap in P. berghei [99]) that are expressed 6. Sex ratios in the parasitophorous vacuole, but are later secreted and all six members seem to form an extracellular multi-protein complex that Although a female gametocyte gives rise to a single gamete and is important for cell–cell interaction of newly emerged female P. a male gametocyte produces eight gametes following gametogen- falciparum gametes. Interestingly one of these proteins PfCCp4 has esis in the mosquito, the sex ratio of gametocytes in the vertebrate been shown to associate with Pfs230 [100]. The CCP proteins are host is very variable. Sex ratio tends to be female biased in all hypothesised to play a role in mediating signalling events during Plasmodium species (reviewed by Carter and Graves) [17].Ithas fertilisation as well as having subsequent roles during development been reported that the sex ratio of P. falciparum gametocytes varies within the mosquito or in the initial gamete–gamete binding event significantly between individual gametocyte carriers and over the [98]. Interestingly a homologue of HAP2, responsible for a non- course of infection. In a study in Cameroon the mean proportion fusion phenotype in gametes of the single cell alga Chlamydomonas, of male to female gametocytes was 0.217 (that is 3.6 females to was shown to be essential for fertilisation (but not initial gamete each male) [89]. However, the mechanism by which sex ratios binding) in P. berghei [101]. It is clear that some of the important 62 D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 players in fertilisation of Plasmodium gametes have being identified targets) to kill these two individual life cycle phases to reduce as well as insight gained regarding their respective roles. the spread of drug resistance. The point has also been well made that the mosquito stages of development are vulnerable in terms of the extremely low parasite numbers and are thus an excel- 8. Cytoadherence and sequestration lent target for drugs (or vaccines) [6]. There is a great deal of early work showing that treatment of patients with antimalarial The parasitophorous vacuole and surrounding pvm are signifi- drugs inhibits subsequent parasite development effectively (e.g. cant barriers to be overcome by proteins that are exported to the inhibitors of dihydrofolate reductase such as pyrimethamine) in red cell cytoplasm and beyond to the red cell surface. It has been the mosquito at the various stages of development following a shown that proteins containing a short conserved sequence motif blood meal (sporontocidal drugs, reviewed by Butcher [59]). How- (referred to as Pexel or HT) near to the N-terminus are able to tra- ever, persuading funders to support translational research that verse the pvm [102,103] in both sexual and asexual parasites. This focuses on transmission-specific drug targets is understandably has been particularly well studied for the family of var gene prod- challenging. With the recent availability of funding to attempt elim- ucts (PfEMP1) involved in cytoadherence of asexual blood stage ination/eradication of malaria, there is a growing recognition of the parasites to endothelial cells during sequestration of parasitised importance of controlling transmission [5]. Screening for new com- red cells within the microvasculature. Little is known about pro- pounds that are active against developing and mature gametocytes tein trafficking and cytoadherence in gametocytes. However, it is is underway in several locations worldwide. known that developing gametocytes also sequester in vivo. Only Most antimalarial schizonticides have no effect on gametocyte mature stage V gametocytes (and probably also sexually commit- development and therefore whilst curing the patient, allow trans- ted ring stage parasites) appear in the circulation. Early reports mission of disease to others in the population for weeks after indicated that the spleen and bone marrow are the predominant clearance of asexual parasites [111]. Furthermore there is evidence sites occupied by developing gametocytes [104]. It has been shown of enhanced rates of transmission of drug resistant parasites from that a particular var gene subset (type C) is transcribed in game- drug-treated individuals [112]. Other drugs such as quinine and tocytes in vitro and that the pattern of expression is independent mepacrine [113] have even been reported to increase gameto- of the PfEMP1 phenotype of the asexual parasites from which cyte numbers or transmission to mosquitoes. Some drugs such as the gametocytes were derived [105]. Up to 40–48 h of develop- chloroquine (up to around day 6 of development) [114] and qui- ment, gametocytes were found to cytoadhere to endothelial cells nine kill early gametocytes due to their effects on degradation of expressing CD36 via PfEMP1 in a similar fashion to asexual par- haemoglobin, but have no effect on the later stages. Atovaquone asites [106]. Later stage gametocytes have been shown adhere to is known to selectively inhibit the parasite ubiquinol oxidation cells expressing ICAM-1 and binding was released by antibodies to site (Q ) of cytochrome b that is involved in the maintenance of three host cell receptors present in human bone marrow and stro- 0 mitochondrial membrane potential [115–117] and is active against mal cell lines [107]. The mechanisms underlying clonal antigenic the younger gametocyte stages [118]. The favourable effects of variation of var gene products in asexual parasites and their role in artemisinin derivative-based combination therapy (ACT) on trans- host immune evasion is well documented elsewhere. Another sub- mission are also well documented [119]. Although artemisinin telomeric multigene family stevor has been hypothesised to have a derivatives can reduce gametocyte carriage [120,121] transmis- role in gametocyte cytoadherence [108]. Subsets of this family are sion can still occur following ACT [122] which may in part expressed in gametocytes and in contrast to var gene expression, reflect the rapid clearance of artemisinin-based compounds. It is the same stevor subset is expressed in the asexual parasites from likely that the reduction in gametocyte numbers following ACT which they are derived [105]. at least partly reflects an indirect effect due to the rapid clear- Reports on the immune response to host red blood cell surface- ance of asexual parasites [123]. Evidence suggests that artemisinin localised gametocyte proteins are more limited, but it has been kills early (sequestered) forms, but not mature gametocytes demonstrated using flow cytometry that antibodies are present in [121,124–127], but more detailed in vitro studies on this would be Gambian children (gametocyte carriers 1–2 weeks following drug worthwhile. treatment) that react with antigens exposed at the infected red cell Primaquine (used widely to treat P. vivax and P. ovale infection) surface of cultured mature P. falciparum (clone) 3D7 gametocytes. and other 8-aminoquinolines can certainly kill mature P. falci- These were distinct from those expressed at the red cell surface of parum gametocytes [59,128–130]. Prior to the introduction of ACT asexual blood stage 3D7 parasites and may have a role in modu- as a first line treatment, a single dose of primaquine was recom- lating transmission since the presence of these antibodies between mended by WHO to reduce transmission in low endemic areas days 7 and 14 correlated with reduced gametocyte densities at day [131]. 8-Aminoquinolines have also been included previously in 28 following drug treatment [109]. Study of these red cell compo- mass drug administration programmes [132]. A single dose of pri- nents during gametocyte development and their interaction with maquine has been used successfully in children in a hypedrendemic host tissues is an important area for future research and may reveal region of Tanzania to clear residual gametocytes following treat- novel means to control transmission. It has also been hypothesised ment with sulphadoxin-pyrimethamine and artesunate [127].It recently that proteins expressed on the red cell surface of game- was suggested that regimes including a single dose of primaquine tocytes may play a role in determining when mature gametocytes could be used to reduce post-treatment infectivity and importantly are released into the circulation and moreover where they go to reduce the likelihood of transmission of drug resistant parasites. maximise their chances of being picked up by a mosquito [110]. The main problem with primaquine is a significant safety issue especially in areas with high levels of glucose-6 phosphate dehy- 9. Drugs that affect gametocyte development and reduce drogenase deficiency where serious haemolytic anaemia can occur transmission in some forms of the condition (reviewed by Vale et al. [130]). The seemingly unique ability of primaquine and its relatives to It could be argued that the ideal antimalarial drug should kill kill mature P. falciparum gametocytes (as well as hypnozoites of both asexual blood stage parasites and gametocytes, thus curing P. vivax and P. ovale) necessitates discovery of its molecular tar- the individual and protecting the population by preventing onward get which, since 8-aminoquinolines have a relatively poor activity transmission of infection. There are also arguments favouring the against asexual parasites, is likely to be a component of one of the combined use of distinct drugs (that inhibit distinct molecular biochemical pathways that are specific to gametocytes (and hyp- D.A. Baker / Molecular & Biochemical Parasitology 172 (2010) 57–65 63 nozoites). Though its mode of action is not known, there is evidence [20] Lasonder E, Ishihama Y, Andersen JS, et al. Analysis of the Plasmod- that it might target mitochondrial function (reviewed by Vale et al. ium falciparum proteome by high-accuracy mass spectrometry. Nature 2002;419:537–42. [130]) and if so this is likely to be some aspect that is of particular [21] Young JA, Fivelman QL, Blair PL, et al. The Plasmodium falciparum sexual devel- importance to maturing gametocytes. opment transcriptome: a microarray analysis using ontology-based pattern identification. Mol Biochem Parasitol 2005;143:67–79. [22] Khan SM, Franke-Fayard B, Mair GR, et al. Proteome analysis of separated 10. Concluding remarks male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell 2005;121:675–87. Though the study of gametocytes has received relatively little [23] Alano P, Read D, Bruce M, et al. 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