The Integral Plasmodium Life Cycle Phenomenon: Gametocyte Genes

The World Health Organization (WHO) has estimated approximately 198 million cases and 5,84,000 annual deaths due to malaria worldwide [1]. The five species of Plasmodium that infect humans most commonly are P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi with P. falciparum the most deadly among them [2]. The malarial parasite life cycle involves two hosts; a malaria-parasite infected female Anopheles mosquito and the human host. During the blood meal transmission from the vector to the vertebrate host is ensured by the sporozoites present in the saliva of the biting vector. In human host, the asexual life cycle begins where some parasites differentiate into sexual erythrocytic stages (gametocytes). The process of development of sexual stages from the asexual stages within the host is called gametocytogenesis. The sporogonic cycle continues in the mosquito when these gametocytes, male (microgametocytes) and female (macrogametocytes) are ingested by an Anopheles mosquito during the blood meal. In the mosquito’s stomach, the microgametes fuse with the macrogametes forming zygotes and later develop into oocysts. The oocysts grow, rupture, and release sporozoites, which reach the mosquito’s salivary glands so that whenever, the infected mosquito bites another human host, inoculation of the sporozoites into a new human host perpetuates the malaria life cycle [3]. Gametocytes are the sexual stages of the malaria parasites that develop in the human host and are transmitted to the definitive vector-mosquito to continue its life cycle. Although it is well known that gametocytes are responsible for malaria transmission, relatively little is known about the gamete biology and the molecular mechanisms involved in the process of gametocytogenesis. The gametocytogenesis is the transition phase in the life cycle of the parasite where it changes morphologically as well as biochemically from asexual to sexual stages which occur in the inner membrane complex (IMC) of the erythrocyte [4]. This sexual differentiation is dependent upon a coordinated cascade of gene expression, involving a number of ‘sexual-stage-specific’ genes and their protein products [5]. Gametocytes are the key targets in the life cycle of the malaria parasite and for eliminating malaria it is necessary that the control strategies aiming to eliminate gametocytes need to be devised.


Introduction
The World Health Organization (WHO) has estimated approximately 198 million cases and 5,84,000 annual deaths due to malaria worldwide [1]. The five species of Plasmodium that infect humans most commonly are P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi with P. falciparum the most deadly among them [2]. The malarial parasite life cycle involves two hosts; a malaria-parasite infected female Anopheles mosquito and the human host. During the blood meal transmission from the vector to the vertebrate host is ensured by the sporozoites present in the saliva of the biting vector. In human host, the asexual life cycle begins where some parasites differentiate into sexual erythrocytic stages (gametocytes). The process of development of sexual stages from the asexual stages within the host is called gametocytogenesis. The sporogonic cycle continues in the mosquito when these gametocytes, male (microgametocytes) and female (macrogametocytes) are ingested by an Anopheles mosquito during the blood meal. In the mosquito's stomach, the microgametes fuse with the macrogametes forming zygotes and later develop into oocysts. The oocysts grow, rupture, and release sporozoites, which reach the mosquito's salivary glands so that whenever, the infected mosquito bites another human host, inoculation of the sporozoites into a new human host perpetuates the malaria life cycle [3]. Gametocytes are the sexual stages of the malaria parasites that develop in the human host and are transmitted to the definitive vector-mosquito to continue its life cycle. Although it is well known that gametocytes are responsible for malaria transmission, relatively little is known about the gamete biology and the molecular mechanisms involved in the process of gametocytogenesis. The gametocytogenesis is the transition phase in the life cycle of the parasite where it changes morphologically as well as biochemically from asexual to sexual stages which occur in the inner membrane complex (IMC) of the erythrocyte [4]. This sexual differentiation is dependent upon a coordinated cascade of gene expression, involving a number of 'sexual-stage-specific' genes and their protein products [5]. Gametocytes are the key targets in the life cycle of the malaria parasite and for eliminating malaria it is necessary that the control strategies aiming to eliminate gametocytes need to be devised.

Gametocyte Development Morphology
The gametocytes are the gamete precursor in the mosquito midgut to the gamete sporogamy. The growth and development of the P. falciparum gametocytes is divided into five (I-V) morphologically distinguishable stages spread over 8-17 days from merozoite invasion to mature gametocyte in the host RBCs [6]. Stage I gametocytes are very much similar to the young asexual trophozoite. The first morphological differentiations start appearing in stage IIb as a new subpellicular cytoskeleton starts to form supported by few microtubules; giving the gametocytes their characteristic elongated shape and pointed ends [5]. The distinguishable male and female forms start to show at stage III where one region of the parasite straightens and the characteristic D-shape of the gametocytes starts to form [5,7]. The gametocytes become more symmetrical in stage IV as the subpellicular cytoskeleton formation is completed [4]. Sexual differentiation is accentuated in female gametocyte at IV stage with a marked increase in the density of ribosomes, endoplasmic reticulum, Golgi complex and mitochondria than in male gametocytes. This clearly indicates the subsequent development of the macro-gametocyte as the fertilized egg. Successful transformation of the stage IV parasite to the morphologically mature stage V gametocyte is accompanied by a collapse of the pointed spindle into a crescent shape where the host cell appears as a flattened thin layer around the parasite [5]. The morphological characteristics of gametocyte developmental stages are described in Table 1.

Localization of Gametocytes
The developing gametocytes are absent from the peripheral circulation, as they adhere to the endothelial erythrocytes of the host organs (heart, lung, liver, brain and tissues as skin) and thus are able to avoid the phagocytic clearance [8]. This is the major reason for practically nil gametocytes to be seen in the peripheral blood and it is only after the peak of asexual parasitaemia that the gametocytes can be seen in peripheral smears. The cytoadherence phenomenon in infected red blood cells (IRBCs), is mediated by a series of host receptors: most strongly with the glycoprotein CD36, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and thrombospondin (TSP) [9][10][11]. The parasite ligand for CD36, ICAM-1 and TSP has been identified as the P. falciparum erythrocyte membrane protein 1 (PfEMP-1) [12]. The parasite ligands associated with stage II and IV gametocyte sequestration in bone marrow include ICAM-1, CD49c, CD166 and CD164 receptors [13,14]. The ligand candidate or their marker could be targeted by immunotherapies that specifically bind or inhibit them. Some of the parasite target domains are listed here: • PfEMP-1 -Present on the parasitized RBCs and form a key role in cytoadherence; the importance of this parasite ligand is well known [15].
• STEVOR -The subtelomeric variant open reading frame (stevor) genes code for the STEVOR proteins found on the surface of gametocyte III-IV stages have a major role in antigenic variation [16].
• RIFIN -Repetitive interspersed family (RIFIN) gene family play a crucial role in antigenic variation and developmental stages of parasites. The rif gene PF13_0006 of this family is most prominently expressed on stage V gametocytes and another rif gene PFI0025c is present in stage II and III gametocytes [17].
Host receptors on parasite ligands serve as potential vaccine targets for promising transmission-blocking vaccines [18].

Gene Expression in Gametocytes
In malaria life cycle gametocytes are the only stage which mediates transmission from the human host to the vector (Anopheles). The start of gametocyte production in the host bloodstream represents a transition period inducing several morphological and biochemical changes. The morphological changes in the gametocyte are accompanied by distinct patterns of sexual stage-specific gene expression [19]. The genes which are majorly involved in the process of gametocytogenesis are viz. Pfs16, Pfs25, Pfg27, Pfs48/45, Pfs230 and Pfg377 ( Table 2). The differential expression of these specific genes help in the intra-erythrocytic development of the parasite and the specific promoter regions in these stage specific genes are essential for the transcriptional activity of these genes [20]. Most of these studies have concentrated on surface antigens, the majority of these characterized antigens are gamete antigens that are synthesized during gametocytogenesis [7,19]. The synthesis of Pfs16 protein, one of the earliest to appear is found in parasitophorous vacuole membrane (PVM) of the young gametocytes marking the onset of gametocyte synthesis [20]. In the gametocyte formation Pfg27/25 is expressed approximately 30 hours after erythrocytic invasion [21]. The presence of Pfs230 and tubulin protein are pre-required for gamete formation and these are most likely the fertilization receptors, where Pfs48/45 though present on both male and female gametes are only essential to male fertility [22]. Several proteins are required for sexual development and most notable proteins found in the mature female gametocyte are Pfs230 and Pfs48/45. The emergence of gametes from the red cell is mediated by osmiophilic bodies containing Pfg377 responsible for disruption of the enveloping RBCs [23]. The signalling processes regulating the formation of microgametes are triggered by   the temperature fall (about 5°C) along with the increased presence of the xanthurenic acid in mosquito [24,25]. The molecular events of the gametocytogenesis are not fully understood yet and it is believed that more genomic and proteomic studies will unravel the molecular processes involved in this phenomenon. The sexual stage specific genes serve as good targets for gene disruption studies as they are responsible for malaria transmission.

Genetics of Gametocyte Genes
The gametocytogenesis is a regulated process marked by distinct morphological and coordinated expression of sexual stage genes. This sexual differentiation process involves the development of gametocytes within the host RBCs from indistinguishable trophozoites (Stage I) to highly specialized, infective mature gametocytes (Stage V).

Pfs16
The occurrence of Pfs16 in gamete preparations primarily is due to the PVM that are still attached to the female gametes. The strains which produce mature gametocytes have high level of Pfs16 at the earliest stage though its role during gametocyte maturation is not well known [26]. The expression of the Pfs16 gene in the sexual committed parasite is one of the earliest events in the sexual differentiation process and represents one of the earliest known markers of an individual parasite's commitment to gametocytogenesis [27]. The promoter activity of Pfs16 can be detected as early as 24 hours after the erythrocyte invasion indicating that Pfs16 is present during entire gametocyte maturation from stages I-V [27]. The early appearance of this gene in abundance is indicative that Pfs16 have a major role to play in the formation of gametocytes. Some studies showed that though no morphological changes were seen in gametocytes but in male mutants exflagellation was not seen and were found to produce non-infective mosquitoes clearly indicating that importance of Pfs16 for optimal mature gametocyte production [28]. Pfs16 is indispensable for male gametocyte ex-flagellation and produces a decisive signalling pathway for the gametocyte maturation process. It has been reported that Pfs16 is not essential for sexual development, but may have a role to play in optimal production of sexual parasites [29]. The Pfs16 gene is upregulated at the onset of the sexual differentiation and the protein produced is localised in PVM and in all derived membranous structures [30]. The correlation between Pfs16 cDNA in the first 40 hours of culture and the production of mature gametocytes is also seen hence, indicating Pfs16 as an early marker of the developing gametocytes and can serve as a potential target of new anti-malarial drugs [31].

Pfs 25
Pfs25 is an early expressed sexual stage protein found on the surface of P. falciparum zygotes and ookinetes [32]. It is a cysteine-rich 25-kDa antigen composed of four tandem epidermal growth factor (EGF)like domains putatively anchored to the parasite's surface through a glycosylphosphatidylinositol (GPI) moiety and mainly expressed after the gametocyte have been transmitted to the mosquito gut [33,34]. Several studies have demonstrated that promoter region (-722 to -308) of Pfs25 is very important for the expression of the gene [34]. The Pfs25 gene used as a marker for female gametocytes, is located in the intracellular vesicles in gametocyte IV and V stages and its presence can be assessed in both non-activated and activated gametocytes [35]. Major part of the Pfs25 coding regions is conserved and recombinant vaccines based on this gene are currently underway which may assist in the control of lethal forms of human malaria [36]. Some studies suggest there is limited sequence polymorphism in Pfs25 and the antibodies against it showed relatively transmission blocking activity against field isolates [37].

Pfg27
Pfg27 is the sexual stage specific phosphoprotein present from the onset of sexual differentiation and is found throughout gametocyte maturation [38,39]. It was demonstrated that Pfg27 defective gametocytes show, distinct abnormalities in intra and extra-cellular membranous compartments, such as accumulation of parasitophorous vacuole-derived vesicle in the erythrocyte cytoplasm, large intracellular vacuole and discontinuities in their trilaminar cell membrane [40]. The antibody recognizes a 15 amino-acid region in the recombinant Pfg27 and also cross-reacts with the reduced and denatured forms of Pfs230 and Pfs48/45, in immunoprecipitation [41]. The protein is essential for the sexual development and maintaining the sexual phenotype of the parasite and its absence results in vacuolated highly disarranged and disintegrating parasites [42].

Pfs230
Another major protein associated with the gamete differentiation is Pfs230, a 360 kDa member of P. falciparum protein family. The Pfs230 gene is the largest of the 10-member protein family found in P. falciparum with seven double cysteine rich domains present on the gamete surface and has a repeated pattern of associated amino acid motifs [43]. Pfs230 is involved in protection of the parasite from the contents of the blood meal, in fertilization, or in the formation of exflagellation centers [44]. When gametogenesis is stimulated by incubating mature gametocytes in human serum at 25°C, Pfs230 undergoes proteolytic lysis from a 360 kDa precursor to a doublet referred to as the 310 kDa to mainly associate with the surface of the newly formed gamete [45]. Using peptide specific antibodies, the amino termini of the 307 and 300 kDa forms have been mapped to between 477-487 and 523-555 amino acid, respectively, which is the region between the glutamate rich repeats and the cysteine motif domains [46]. These studies reveal that Pfs230 is the surface molecule on males that mediates RBC binding and plays an important role in oocyst development, a critical step in malaria transmission [47]. It has been observed that Pfs230-minus males, in the presence or absence of Pfs48/45, are unable to undergo exflagellation; also oocyst production and mosquito infectivity is significantly reduced, about 96-92% and 76-71% respectively [48]. It was found that only the processed forms exposed on the surface of the gamete are the targets of antibodies that block parasite transmission to the mosquito [49]. This mainly confirms the potential of Pfs230 as a prime target for malaria transmission blocking reagents and contributes to the understanding of the molecular mechanisms involved in early stages of P. falciparum development in the mosquito midgut.

Pfs 48/45
Pfs48/45 is specifically expressed on the surface of the macrogametes of the malaria parasites and is a well-established transmission-blocking (TB) vaccine candidate [50,51]. Pfs48/45 consists of 1.5 double domains belonging to a family of proteins that are characterized by six positionally conserved cysteines (6-Cys proteins) having a signal peptide with putative GPI anchor sequence to locate on the parasite surface [52,53,22]. This sexual stage-specific Pfs48/45 plays a central role in male fertility and assists in male micro-gametes attachment to fertile female macrogametes [50]. The presence of this gene is proven in sexual stages but is also present in asexual stages though in very small percentages [54]. The Pfs48/45 which is a target for developing transmission-blocking vaccine proves difficult for vaccine construction because of the conformational nature of its epitopes as shown by the panel of monoclonal antibodies (mAbs) developed from murine and rat could recognize only five different epitopes [50].

Pfg 377
The stage IV gametocytes show the presence of osmiophilic bodies, electron dense organelles observed in ultrastructural studies, located preferentially beneath the surface of female gametocytes [55]. The mature stage V macrogametes emerge only in the mosquito midgut from the RBC and their emergence is facilitated by the presence of osmophilic bodies in gametocytes. The protein which is associated with osmophilic bodies is encoded by Pfg377 gene [56]. The Pfg377 gene located on chromosome 12 is single exon of 9360 bp and a novel sexual stage antigen of P. falciparum. It is highly hydrophilic, 377 kDa protein and homologous of which is found in human, rodent, avian and primate species. Pfg377 shows a characteristic granular appearance due to the shape and the cellular location of the osmophilic bodies. It contains four regions characterized by the presence of repeated amino-acid motifs. Region 1 is a proline-rich region beginning at amino acid residue 99, region 2 is a glutamic acid-rich region at residue 757, region 3 is a histidine-rich region at residue 866, and region 4 is a histidine-and glutamine-rich region at residue 1900 [56]. Pfg377 contains repeated sequences of highly variable length among different parasite clones, thus indicating the presence of more than one parasite clone. Recently it has been shown that targeted disruption of Pfg377 gene results in reduced emergence efficiency of gametocytes from the erythrocyte and the Pfg377 negative gametocytes also resulted in no infections completely [57].

Discussion
Malaria caused by the protozoan Plasmodium parasite comprises of a complex life cycle having infective stages; (pre-erythrocytic and erythrocytic phases) and a sexual phase known as gametocytogenesis (gametogenesis and sporogony stages). In this review article we have attempted to re-assess the knowledge regarding the gametocyte specific gene expression during the various stage developments. Malaria infections in symptomatic and asymptomatic episodes are most frequent in the transmission season, usually when mosquitoes are most abundant. The role gametocytes play in transmission of the diseases is highly pronounced but the molecular processes which are totally responsible for the process are still not fully known.
The dynamics of gametocytes are understood better extensively for symptomatic malaria cases, where waves of asexual parasitemia and fever are accompanied followed by increases in gametocyte prevalence [58,59]. The Plasmodium parasites know exactly at which point in their complex life cycle a particular gene expression is needed. Gametocytespecific proteins highly represented in gametocyte proteome are surface proteins which include; gamete antigen Pfg27 protein, Pfg377 protein, sexual stage specific protein Pfs16 and transmission-blocking antigen proteins: Pfs48/45 and Pfs230 [60]. Pfs48/45 and Pfs230 are located as a complex on the plasma membrane of the gametocyte with only Pfs48/45 being directly anchored to the membrane by a GPI moiety.
The gametocyte gene expression profiling approaches need to be coupled with more detailed knowledge of the underlying epidemiology dynamics of gametocyte carriage and infectivity. The success of malaria control and elimination is dependent on our understanding of gametocytes which in turn will result in a huge step in the right direction for developing the transmission blocking vaccine.