Cellular and Molecular Biology of Plasmodium Parasites

Abstract

Understanding the cellular and molecular biology of any infectious agent is the mainstay of its successful prevention and control. Therefore, in this review, various aspects of the cellular and molecular biology of the Plasmodium parasite were critically reviewed. Plasmodium parasite is characterized by the presence of three different invasive forms (Sporozoites, Merozoites, and Ookine/Oocyte), which are morphologically and genetically distinct. The size of the Plasmodium genome, which comprises nuclear, plastid, and mitochondrial genomes ranges in size from 20 to 35 megabase (Mb) with 23 million bases, which translate into 7132 genes in Plasmodium ovale and 5507 in Plasmodium falciparum. Plasmodium species are found to be the most AT-rich genome (80%), and their GC% contents are merely less than 20%. Gametocytogenesis indicates the commencement of the sexual development, which is regulated by signal transduction and expression of genes such as Pfs16, Pf14.744, Pf14.748, Pfpeg3/mdv1, and Pfpeg4. In these stages, only 20% of all Plasmodial genes are expressed.

1. Introduction

The Plasmodium parasite is an associate of the Apicomplexa, a broader phylum of obligate intracellular parasites, all of which are eukaryotic [1]. As a result, the cellular and molecular biology of the parasite is comparable to that of other eukaryotes, and its intracellular lifestyle is one of its fundamental and distinctive characteristics. The parasite has a close bond with its host cell that can be explained at the cellular and molecular levels due to its intracellular existence. The malaria parasite has three distinct invasive morphological forms: Ookinete (only in the vector), Sporozoites (in the vector and human host), and Merozoites (in erythrocytes). Female Anopheles mosquitoes bite and inject Sporozoite forms, which are quickly cleared from the bloodstream, with many of them moving to the liver and invading hepatocyte cells [2]. As they advance to produce Merozoite (exoerythrocytic stage) forms, which are later freed into the bloodstream, the intracellular parasite radically modifies the host-infected red blood cell as it grows and replicates, resulting in an inflexible and ill-disfigured blood cell. The main consequences of malaria infection, which include fever, anemia, lactic acidosis, and, in some severe conditions, unconsciousness and loss of life, are mainly caused by these alterations [3]. This is because the parasite takes almost the overall control of the physiological activities of the infected erythrocyte, which is later destroyed.

Therefore, this review aims to describe the biology of Plasmodium species at the cellular and molecular level. This is because a better understanding of this baseline information on the parasite may reveal a very realistic approach toward the control and prevention of malaria infection through the use of some parasite’s parasite-specific proteins, especially for the production of malaria vaccine.

2. Invasive form of Plasmodium species

2.1 Sporozoite

The process known as sporogony, which results in the generation of thousands of infectious Sporozoites, starts when a motile Ookinete is stopped between the midgut epithelium and the surrounding basal lamina of the vector [4]. According to their morphology, Sporozoites are 10–15 m long and elongated, resembling worms. The multinucleate Oocyst in the female Anopheles mosquito’s midgut develops into Sporozoites, which are the parasite’s infectious form for the human host and other vertebrate host cells [5, 6]. Thus, the female Anopheles mosquito inoculates the Sporozoite into the susceptible host to begin the malaria infection. Due to the requirement that they infiltrate one target organ in each host during their infectious phase, Sporozoites are distinct from other Plasmodium invasive stages. The vector (female Anopheles mosquito) deposits about 100 infective stages of the parasite (Sporozoites) in the skin in the process of taking a blood meal. Once injected into a mammalian host, [7, 8], in 20–60 seconds, these Sporozoites quickly move by gliding locomotion with the aid of several proteins, such as CircumSporozoite protein (CSP) and Egress Cysteine Protease-1 (ECP1) [5], which allow them to cross cell membranes and enter the host cell. Sporozoites initially penetrate the sinusoidal barrier after entering the liver by blood circulation, then they continue through and eventually invade hepatocytes [9, 10]. To prevent lysosomal breakdown, Sporozoites are housed in a parasitophorous vacuole in the hepatocyte [11]. The potential of Plasmodium falciparum Sporozoites to exploit a range of invasion mechanisms, including the active involvement of various parasite proteins that are currently unknown and uncharacterized, may be the cause of this ability to infiltrate a variety of target cells [12].

During the erythrocytic cycle, the parasite exits in three distinct morphological phases, the ring stage lasting roughly 24 hours in P. falciparum, accounting for about half of the intraerythrocytic cycle but being metabolically nondescript [13]. While in the hepatocyte, they develop, multiply, and differentiate into Merozoites in about 7–10 days before they burst and invade the erythrocyte. Finally, during the Schizont stage, parasites go through 4–5 cycles of binary division, creating Merozoites that burst from the host cell to infect fresh erythrocytes and start a new round of infection [14].

According to its structural design, Sporozoites have micronemes, rhoptries, and dense granules at their posterior ends. These organelles produce several adhesins and invasins that are crucial for attachment to and penetration of the host cell (erythrocyte) [8].

2.2 Merozoite

This is an asexual erythrocytic stage of Plasmodium, and in terms of size, it is smallest compared to other developmental stages of the parasite. It is considered to be the smallest known eukaryotic cell, measuring only about 1–2 μm, and is well suited for erythrocyte colonization [15]. It is oval-shaped and contains some organelles that are secretory in nature and release certain specific proteins necessary for the invasion of new red erythrocytes without lysing them [16, 17]. Some proteins, such as Merozoite surface proteins (MSP) (MSP-1, MSP-2, MSP-3, MSP-6, MSP-7), are some of the proteins that cover the Merozoite’s outer membrane from the outside, giving the Merozoite a fuzzy appearance [18].

Due to the formation and secretion of a special protective sheath called the parasitiphorous vacuole (PV) and the membrane that delimits the parasitiphorous vacoule, Plasmodium can avoid the host’s immune system, which is one of the main benefits of the Merozoites living inside the cells of its host [19].

Merozoites, have micronemes, rhopteries at the apical end, and dense granules, such as Sporozoites. These organelles perform a very crucial role in the interaction between ligands and receptors during the Merozoite’s invasion of erythrocytic cells with the aid of a protein on its surface called merozoite surface protein-1 (MSP-1).

2.3 Oocyte and Ookinete

The only diploid (2n) form of the parasite, called Ookinete, is invasive, mobile, and only forms after successful gametogenesis and fertilization in blood bolus in the midgut of the vector. To invade the stomach epithelial cells and avoid digestion by crossing the midgut wall, newly developed Ookinetes migrate via the of the alimentary bolus, pass through the protective layer, and build up a microvillar network in the peritrophic matrix [12, 20]. According to Li et al. [21], Ookinete colonization of midgut epithelia is a critical phase in the establishment and progress of the parasite in the mosquito, and it is also a key process in the transmission of malaria. After which, it bonds to the basal lamina, loses its kinesis (mobility), and develops into an Oocyst, a trophic form of the parasite that may reproduce. Numerous Sporozoites formed from the multinucleated Oocysts are freed into the fluid medium (hemolymph) [22], where they passively drift before actively invading salivary glands. These Sporozoites remain viable and active but do not replicate, but instead grow and mature to the infectious stages that are subsequently transmitted to the next mammalian host [23].

3. Plasmodium genome

The genetic makeup of the malaria parasite is complex, but with the help of modern molecular technologies, it is possible to analyze the genetic material and create valuable data. Most developmental stages (Meroziotes and Sporozoites,) of Plasmodium species exist as haploid (n) (that is containing only seven chromosomes) throughout their life cycle, except for a brief diploid (2n) phase that occurs after the fusion of macrogametocyte (female) and microgametocyte (male) in the midgut of the mosquito. According to Su et al. [24], the genomes of various Plasmodium species range in size from 20 to 35 megabases (Mb), which is by far greater than that of Yeast cells (Saccharomyces cerevisiae). The 23 million base pair Plasmodium genome has 7132 genes in P. ovale and 5507 in P. falciparum [25]. The genome is distinctly divided into nuclear, plastid, and mitochondrial genomes. The first two genomes (nuclear and plastid) have genome sizes of 35 and 6 kilobases, respectively, the nuclear genome, which has 14 chromosomes, is the largest of the three [26]. The parasite has seven encoding loci and one 18S (18s-5.8s-28s) ribosomal RNA (rRNA) on almost all the fourteen (14) chromosomes, S-type r-RNA-producing genes are found in the female Anopheles mosquito (vector), whereas A-type genes are found largely inside the secondary host (humans) [27].

The shotgun-sequencing technique was used in the year 2002 to produce the first complete genome of Plasmodium 3D7 (a laboratory-adapted strain), where the result indicated that the parasite contained a high (80.6%) proportion of A (Adenine) + T (thymine) composition. This makes Plasmodium to be the most AT-rich genomes known to date [28]. Extended sequences of adenines (As), thymines (Ts), and thymine-adenines (T-A) are found in introns, intergenic, and centromeric regions (up to 99% A + T content), particularly in subtelomeric regions, all these contribute to the genome’s complexity. Additionally, it is crucial to comprehend the molecular biology of Plasmodium falciparum not only because of its unicellular and intracellular mode of life that causes the most severe type of human malaria but it also harbors the nuclear genome with the least amount of GCs. In fact, according to Nikbakht et al. [29], its GC% is merely less than 20%. The majority of next-generation sequencing (NGS) technologies have struggled to accurately sequence P. falciparum DNA as a result of these unique traits, and it has been proposed that using the existing 3D7 genome sequence as a standard for clinical isolates leads to inaccurate estimates of genetic diversity [30]. One of the most distinctive characteristics of the P. falciparum genome is the lack of intron splicing, which results in a weak association between the length of a gene and its functional domains [31].

Around telomeres, three well-known gene families—VAR, RIF, and STEVOR—are clustered and responsible for immune evasion. The proteins that each of the genes codes for are Pf EMP1 (P. falciparum Erythrocyte Membrane Protein 1), RIFIN (Repetitive Interspersed Family), and STEVOR (Subtelomeric Variable Open Reading Frame) respectively. Cytoadherence, which isolates cells and leads to generous infection, is facilitated by PfEPM1. The Pf EMP1 protein family, which is encoded by around 60 different genes, is only present in the early gametocyte stage. These proteins are kept in the knobs on the surface of infected red blood cells and join host receptors in the early gametocyte stage. Later stages of cytoadherence seem to involve a host receptor response. Inflammation, transmission, chronic infection, and pathogenesis are predominantly caused by the VAR gene family, and the generation of protective immunity while RIFINS modify the antigenic surface of infected red cells. Forty to fifty STEVOR genes code for stevor proteins, and they exist on Maurer’s cleft and reach their climax 28 hours after the invasion. In stage III, they are delivered to infected erythrocytes after being expressed in stage I [32].

4. Biology of gametocyte

The intriguing gametocyte stage, a specialized sexual precursor, is in charge of transmitting the malaria parasite from humans to anopheline mosquitoes. Its consideration goes beyond biology and may affect case management, transmission control, and preventing the transmission and the spread of parasites that are resistant to treatment. The cellular growth, metabolism, and gene expression patterns of P. falciparumgametocytes differ significantly from those of their asexual precursors; in addition, they also differ in terms of morphology and functions [33].

The gametocytes’ half-lives have been estimated to be between two and three days, but some have been seen to last for as long as four weeks. One of the most conspicuous morphological characteristics of gametocytes is their crescent form appearance due to the presence of a sub-pellicular microtubule-based cytoskeleton and the surrounding double membrane. Two hundred and fifty to three hundred (250–300) genes are selectively elevated at the mRNA level during gametocyte development according to transcriptome analysis. More than 900 proteins are discovered in the P. falciparum proteome, 315 of which are only present in gametocytes [34]. According to Demanga et al. [35], it is possible to stop the growth of gametocytes in red blood cells or stop their fusion in the mosquito’s gut, which will stop the spread of infection to the human host.

4.1 Gametocytogenesis (production of gametocytes)

A primary host (mosquito) and a secondary host (vertebrate host) serve as the two main components of the Plasmodium life cycle. The parasite undergoes asexual replication in the human host after being infected by a malarial Sporozoite, first in the liver (pre-erythrocytic schizogony), then in the erythrocytes (schizogony). The commencement of sexual development is marked by gametocytogenesis, the process by which some asexual parasites irrevocably decide to differentiate into female (macrogametocytes) and male gametocytes (microgametocytes). According to Liu and Miao, for a parasite to be transmitted from a human host to a female Anopheles mosquito, gametocytes are required.

The shift from the asexual to sexual stages is probably regulated by signal transduction and gene expression. A few examples of stimuli that have been shown to trigger the transition to sexual development include parasite lysates, conditioned media, antimalarial drugs, antibodies, temperature changes, pH shifts, and chemicals, such as cAMP and berenil [36]. It also requires specialized parasite stages the gametocytes and Sporozoites [37]. Beginning with gametocytogenesis, the parasite transitions morphologically and biochemically from an asexual life of reproduction inside the human host to a sexual life of development inside the mosquito vector.

The production and export of the parasite protein Pf gametocyte exported protein-5 (PfGEXP5) in the cytoplasm of red blood cells as early as 14 hours after red blood cell invasion is the first indication of gametocytogenesis in both male and female gametocytes. Given that PfGEXP5 expression is PfAP2-G independent and that a limited gametocyte transcriptional program is also initiated in a naturally occurring PfAP2-G mutant parasite line, and additional PfAP2-G independent mechanisms may be involved in the initiation and/or progression of sexual differentiation. Pfs16, pf14.744, pf14.748, pfpeg3/mdv1, and pfpeg4 are a few of the genes expressed at the beginning of gametocytogenesis that may regulate the cellular modifications necessary for early sexual differentiation. The oldest of them, Pfs16, is an aspect of gametocytogenesis that may be seen 24 hours following Merozoite invasion and persists throughout gametocyte growth. Interestingly, at the start of sexual differentiation, the parasitophorous vacuolar membrane (PVM) is where all of the aforementioned proteins are produced. Pfs16 disruption resulted in decreased gametocyte production and mosquito transmissibility [38].

The malaria parasite, despite briefly diploid following zygote formation in the mosquito, has a haploid genome during most of its life cycle. The parasite genome lacks any known sex-specific chromosomes, thus parasite lines produced from a single cell (clone) can produce both male and female gametocytes. Intraerythrocytic parasites can either reproduce asexually within the human host or differentiate into a single male or female gametocyte. Following an asexual reproduction cycle, the malaria parasite has three options: either continue the cycle, grow into a microgametocyte (male), or microgametocyte (female) gametocyte. Gametocyte formation takes longer than asexual schizogony, for example, 26 hours instead of 22.5 hours in P. berghei or, in the more severe instance, 8–12 days instead of 48 hours in P. falciparum. The majority of Merozoites only undergo gametocytogenesis with each round of asexual replication, indicating that the parasite invests surprisingly little in transmission within the mammalian host. In the context of ongoing attempts to eradicate malaria, and adhere gametocytes are a desirable target for intervention [39]. There are several theories put forth to explain this restriction in its reproduction tactic. It may lessen the virulence that vectors experience by ensuring that only a small number of gametocytes are taken up with any blood meal, prevent hosts from developing gametocyte-specific immunity, which would limit transmission, or be the best course of action in the case of competition between co-infecting genotypes, where most resources are devoted to out-competing co-specifics via asexual replication to ensure future transmission [40].

Gametocytogenesis begins roughly 7–15 days after parasites first present in human blood [41]. Gametocytogenesis is the process that creates a gametocyte, which is the only stage of transmission from a human to a mosquito. Additionally, it permits sexual maturation and consequently recombination with other genotypes [42]. This suggests that the G0 phase of the cell cycle is where mature gametocytes are stopped. RNA synthesis can also continue until the sixth day of gametocyte development when it comes to mature gametocytes [38, 43]. This does not take place until shortly before exflagellation and after activation in the mosquito midgut. Commitment, prestige I, and gametocyte development are the three main stages of P. falciparum gametocytogenesis. Early studies have shown that every Merozoite from a single Schizont either commits to sexual development and generates only male or female gametocytes or that every Merozoite from a single Schizont follows the sexual path. About 200–300 mRNAs are selectively expressed or expressed more often in the parasite’s sexual forms during the tightly regulated gene expression program that goes along with sexual differentiation [44].

Gametocyte commitment is the transition from the asexual blood stage to the gametocyte [45]. Expression of the master regulator PfAP2-G, a transcription factor of the ApiAp2 family, is a marker for commitment and is described as a cell state that leads inexorably to later sexual conversion [39]. The DNA-binding protein AP2-G, which is a member of the ApiAP2 family, is a crucial sexual commitment regulator. Forward and reverse genetic analysis of the parasites Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii has revealed that AP2-G is the main regulator of gametocytogenesis. The development of asexual parasites will continue, whereas those that express AP2-G can commit and start gametocytogenesis. The majority of cells do not make AP2-G because H3K9me3, heterochromatin protein 1(HP1), and Histone Deacetylase 2 epigenetically mute it in P. falciparum [46].

Several environmental factors, as well as genetic variables, such as the metabolic and physiological state of the parasite and the host erythrocyte, affect the decision to commit to sexual development. One more environmental factor that promotes commitment to gametocytogenesis is the presence of juvenile erythrocytes [43]. Additionally, there is no doubt that several additional events during the infection have an impact on gametocyte development. The preponderance of the data suggests that individual Schizonts produce Merozoites that are all dedicated to sexual or asexual development with a commitment to sexual development thought to occur in the generation before gametocytogenesis. Additionally, it appears that Schizonts committed to sexual reproduction create gametocytes of the same sex in a mutually exclusive manner. The majority of the information also indicates that the commitment to gametocytogenesis takes place during the late Schizont or early ring stage of the parasite’s life cycle [42].

Prestage I development starts once the parasite commits to gametocytogenesis. After being liberated from the host red blood cell, the sexually committed Merozoites made by the committed parasite during sexual schizogony penetrate freshly formed erythrocytes to form a sexually committed ring. This committed ring-stage parasite transforms into a stage I gametocyte in the succeeding 24–30 hours, which is morphologically and molecularly distinct. Approximately 5–7 days after the stage I gametocyte is formed, it undergoes a maturation process that results in a mature stage V female or male gametocyte [47]. The creation of a pellicular complex beneath the gametocyte plasma membrane in late stage I, which gives the gametocyte its crescent shape, is a significant morphological feature of developing gametocytes [48].

According to a commonly used classification scheme [42], the complex P. falciparum gametocyte developmental phases may be neatly divided into five morphologically distinct stages (I–V). The crescent-shaped stage II gametocyte progressively acquires the elongated morphology that is unique to gametocytes in this species about day 2 of maturation [49]. Compact circular forms with hemozoin are the first gametocyte stages of P. falciparum that have been identified. Stages I, II, III, and IV are in deep tissue where they mature into sausage-shaped stage V gametocytes and resurface in the blood infective for mosquitoes. These stages (I–IV) are completely absent from blood circulation. Mature P. falciparum gametocytes have a density of 100 gametocytes/mL of blood in peripheral circulation and are frequently present at submicroscopic levels. Mature P. vivax gametocytes are larger, rounder, almost fill the stippled red blood cell (RBC), and have a more pronounced nucleus than their P. falciparum counterparts [50].

Major physiological and metabolic changes occur together with dramatic morphological changes during gametocytogenesis. Young gametocytes break down hemoglobin to produce amino acids for protein synthesis, just like asexual stages. Hemoglobin digestion stops in stage III–IV gametocytes [51, 52].

Because infected erythrocyte attachment to microvascular endothelium of various organs and tissues, including the heart, lung, liver, skin, and brain, the mature asexual stages of P. falciparum are not present in the peripheral circulation. During development, sequestration enables the spleen to resist phagocytic clearance. When gametocytes grow, a similar situation arises: stages I to IV are primarily retained in the spleen and bone marrow, while stages V are released into the peripheral circulation and only become infectious to mosquitoes after a further two or three days of circulation [41, 53]. However, studies have shown that early stage II gametocytes and stage I gametocytes have knobs and cytoadhere to CD36 and ICAM-1 on RBC receptors. Research on adhesion has so far yielded a variety of findings. On later stage II to IV gametocytes, however, knobs have never been seen, and binding has been linked to several receptors, including ICAM-1, CD49c, CD166, and CD164. Although PfEMP1 appears to be produced at low levels during early sexual development and subtelomeric variable open reading frame (STEVOR) has been found on the surface of gametocyte-infected RBCs, the parasite ligands implicated in gametocyte adherence have not yet been identified [54].

There is strong evidence that this transition to sexual development, which is necessary for the mosquito vector to transmit the malaria infection, is susceptible to environmental signals. It has been demonstrated that a range of circumstances can stimulate gametocytogenesis both in vitro and in vivo. Sublethal doses of DNA synthesis inhibitors host immune sera, erythrocyte lysate, and a few antimalarial drugs, most notably Chloroquine, are among them. Higher parasite densities, the inclusion of human serum, lymphocytes, reticulocytes, or antimalarial drugs, as well as the presence of soluble components from parasite-conditioned media can all cause an increase in gametocyte formation. The development of P. falciparum gametocytes is also triggered by hemolysis of infected erythrocytes. Gametocyte production is also increased when the malaria parasite is treated with medications, such as steroid hormones, Fansidar, Chloroquine, or Sulfadoxine-Pyrimethamine. Increased gametocyte production in infections that occur naturally can be attributed to any of these reasons. Additionally, the development of gametocytes from asexual parasite precursors is impacted by the human host’s naturally acquired immunity. Increased gametocytogenesis has been linked to several additional circumstances that are detrimental to asexual development or parasite survival [34]. A crucial morphological indicator of growing gametocytes is the crescent-shaped pellicular complex that emerges in late stage I and is made up of a gametocyte plasma membrane, an inner pellicular membrane vacuole, and microtubules [38].

Activation of previously exported effector proteins or new export can cause stage III–V gametocytes to experience numerous substantial modifications to the host cell during gametocytogenesis. One of these adaptations is the change in deformability that occurs as gametocytes progress from stage IV to stage V. It has been established that the exported STEVOR family proteins play a critical role in mediating host cell stiffness. Stage IV’s enhanced deformability may be due to stage V STEVOR internalization, which takes place, while it is still attached to the RBC membrane in stage IV. It may be necessary to make adjustments to the host cytoskeleton network, including spectrin and band 3, similar to the reported deformability switch during gametocyte development [48].

4.2 Gametocytes protein specific

Due to significant changes in cell design, function, and environment, the majority of Plasmodium genes are only activated in a narrow window throughout different stages of the Plasmodium falciparum life cycle [55]. In the sexual stages, 20% of all Plasmodial genes are particularly presented. In P. falciparum gametocytes, the proteins are present in varying concentrations during five different embryonic stages. The majority of these proteins are involved in gametocyte growth, integrity, and fertilization-related processes. Currently, 226 proteins in Trophozoites and Schizonts, 315 in gametocytes, and 97 in Gametes have been identified. In total, 575 proteins (gametocytes plus gametes) are only discovered in sexual phases, while 488 proteins are discovered in both asexual and sexual stages [56].

Additionally, gender-specific proteins are produced later to support gametes during fertilization in the mosquito’s midgut. Male-specific proteins made up 36% of the male gametocyte proteome, while female-specific proteins made up 19% of the female gametocyte proteome. Only 69 proteins are shared by these species, emphasizing how unique each evolutionary form is. Male gametocytes exhibit the most unique proteome of all the P. falciparum lifecycle stages, containing many proteins essential for flagellar movement and rapid genome replication [57].

However, during the sexual stages, a special Plasmodium gene superfamily that encodes proteins with shared six-cysteine domains is produced. Pfs230, Pfs48/45, Pfs230p, Pfs47, P52, P36, Pf41, Pf38, Pf12, P12p, Pf92, and sequestrin16 are the 12 unique members of the Pfs48/45 family. Pfs230, Pfs48/45, and Pfs47 are three of these proteins that are essential for the growth of the parasite, and Pfs48/45 is one of the well-studied, sexual stage-specific surface antigens [56]. All Plasmodium species share the six-cysteine family, which is characterized by largely conserved, 350 amino acid-long double domains that are cysteine-rich and contribute to the tertiary structure of the proteins. Some of these proteins, which are mostly found on the parasite’s surface, are known to be involved in cell-cell communication. The reproductive process and other essential aspects of parasite life cycles depend on the immunogenic proteins Pfs48/45, Pfs47, Pfs230, and Pfs25 [58].

These proteins can be divided into two categories: pre- and postfertilization antigens. Pfs48/45, Pfs47, and Pfs230 are a few pre-fertilization proteins (antigens) that are expressed on the surface of malaria parasite gametocytes and gametes. On the zygote and Ookinete surfaces exist postfertilization proteins that have demonstrated significant immunogenicity with minimal antigenic polymorphism, such as Pfs25. The early stage I-II gametocytes express crucial proteins, such as Pfs16, Pfg27 and the six-coagulation factor C-like (LCCL)-domain-containing PfCCp proteins [59]. Pfs16 is widely distributed throughout the gametocytes’ development [60], and it plays a role in the development and/or stability of the flagellar central apparatus and is crucial to the biology of the male gamete flagellum. However, Pf16 gene disruption does not result in complete infertility. Instead, it produces abnormal flagellar motility and lower fertility [61].

Six-cys proteins Pfs48/45 are well-known sexual-stage proteins that start at step II of the second stage. The Plasmodium Ps48/45 proteins, which are expressed by both male and female gametocytes/gametes during the parasite’s maturation process, are pre-fertilization proteins (antigens). These proteins are members of a family that all Plasmodium species share, but they also stand out due to the presence of domains made up of six cysteine (Cys) amino acid residues, which combine to form one to three disulfide bridges to create a specific tertiary structure [62]. Male fertility depends on the Pfs48/45 protein, which is expressed on the gametocyte surface membrane in P. falciparum. Additionally, individuals in endemic regions need Pfs48/45 to develop high antibody titers that can reduce Ookinete formation and start a transmission-blocking response. Currently, it is believed to be a potential target for the development of a vaccine that stops transmission. Pfs48/45 is difficult to express as a recombinant product because it has a high concentration of Cys residues similar to other proteins expressed in sexual forms [63]. Plasmodium falciparum Pfs48/45 and Pfs230 are sexual-stage antigens that are expressed on the intraerythrocytic surface of gametocytes and emerge on extracellular gametes in the midgut of the mosquito. Within 10 minutes of a mosquito consuming an infected erythrocyte containing gametocytes, the parasite emerges as an extracellular gamete. Surface antigens, such as Pfs48/45 and Pfs230, are exposed to antibodies in the human blood meal by emergence. Both antigens initiate an immune response during a natural infection, and Pfs48/45 or Pfs230-specific antibodies have been shown to stop transmission to mosquitoes [64].

On chromosome 2 lies the 363 kDa (3135 amino acids) protein known as Pfs230. It is a potent antigen of the vaccination that stops the spread of malaria. It takes part in the fertilization of macrogametocytes by microgametocytes. For interaction with erythrocytes and the development of the exflagellation center in male gametes, the Pfs230 gene is necessary. Female gametocytes and gametes express the sex-specific protein Pfs47 on their surface. According to Pfs47 studies, it is not necessary for female fertility. Pfs25 is a protein that is only found on the zygote and Ookinete surfaces of the mosquito host. The parasite that had its Pfs47 gene specifically disrupted created the predicted number of Oocysts. This 25 kDa protein is necessary for the parasite to survive and thrive in the mosquito’s midgut. Inhibition of Ookinete penetration into midgut epithelial cells is caused by loss of P25 antigen [65, 66].

Tubulin, Pfg377, Pfmdv-1, and actin proteins are among the additional proteins. Both sexes of P. falciparum gametocytes express α-tubulin II as early as stage I, but from stage IV onward, the number of female gametocytes noticeably reduced. Pfg377, a protein that is only found in females, is expressed starting in early stage III gametocytes [60]. Actin-1 (pfact1) and Actin-2 (pfact2) are two actin genes that are encoded by the P. falciparum genome, while PfACT2 is only present in the mosquito and sexually transmittable stages and PfACT1 is ubiquitously expressed throughout the whole life cycle. Male gametocytes are normally inhibited from exflagellating when the PfACT2 gene is lost, and it may produce lengthy filaments similar to canonical activities [67]. Up until stage V, the Pfmdv-1 protein, often referred to as Pfpeg3, exhibits significant levels of expression before declining. Although interruption of the Pfmdv-1 expressing gene results in a 20-fold reduction in the production of male gametocytes, the function of Pfmdv-1 is yet unknown [36].

4.3 Gametogenesis and fertilization

For the Plasmodium parasite to spread from an infected person to a mosquito, at least one male and one female gametocyte must be ingested in a blood meal [68]. Gametocytes downregulate some proteins involved in asexual parasite replication and up-regulate others that are involved in sexual development to develop in the mosquito [69]. Each Plasmodium transmission cycle requires one sexual recombination. Even while infected vertebrate hosts’ blood contains mature sexual stages or gametocytes, mating only takes place in the gut lumen of vector mosquitoes. Gametogenesis starts as soon as infected blood enters the intestine and lasts for around 20 minutes. The parasite disintegrates the two erythrocyte membranes that surround it during that brief period [70]. Shortly after being consumed, gametocytes in the mosquito’s blood meal undergo gametogenesis to produce male and female gametes. When the genome is duplicated three times, eight flagellated microgametes are produced in male gametocytes in less than ten minutes. Exflagellation, or the bursting out of the male gametes, is an extraordinary event that allowed Alphonse Laveran to make the groundbreaking discovery of the malaria parasite in 1880. This suggests that mature gametocytes are stopped in the G0 phase of the cell cycle since it only occurs immediately before exflagellation and only after activation in the mosquito midgut. A mature gametocyte is produced after RNA synthesis, which can last up to the sixth day of gametocyte growth. Gametes separate into male and female variants as a result of a drop in temperature, rise in pH, and concentration of Xanthurenic acid [50].

The P. falciparum microgametes and macrogametes emerge from the consumed RBCs in the gut of the mosquito, where fertilization takes place and a motile Ookinete is created. The Ookinete grows into an Oocyst that contains Sporozoites, which travel to the salivary glands of the mosquito after about 10 days to reach full maturity. When the mosquito consumes blood, these adult Sporozoites infect another human host during a blood meal [35].

The male gamete has several stages throughout its incredibly brief (30–40 minute) lifetime. Exflagellation, which involves eight male gametes from each paternal cell budding, is the first stage. To complete cytokinesis and release the gametes, exflagellation requires vigorous flagellar pounding. Exflagellation is followed by free swimming, which involves both fast (5 beats/sec) and slow (1 beat/sec) flagellar beats. Male gametes that are free-swimming come into contact with females and adhere, adhering and ‘rubbing’ on the female’s surface. The final step of gamete fusion is marked by a period of strong flagellar beating that continues even after the male axoneme and nucleus have reached the female cytoplasm [53].

4.4 Sex determination in Plasmodium gametocytes

Sex chromosomes do not affect whether a Plasmodium gametocyte develops into a male or female gametocyte because a single haploid parasite can produce both sexes. Since differential gene expression is shown over the entire genome, sexual dimorphism in Plasmodium is only caused by this. In Plasmodium, the gametocyte sex ratio normally favors females. Male gametocytes can create up to eight gametes, but female gametocytes can only produce one gamete at a time. A female-biased sex ratio would be advantageous to improve fertilization in the mosquito midgut, and therefore promote transmission. However, the gametocyte sex ratio fluctuates during an infection across clones, between regions, and even between particular patients bearing gametocytes [71]. The malaria parasite produces a higher proportion of devoted female Schizonts than committed male Schizonts, which results in the characteristically female-biased sex ratio seen in the parasite.

When it comes to P. falciparum, differentiation into male and female is centered on significant variations in the transcriptomes and proteomes of the sexes. These differences are responsible for the divergent physiology of male and female gametocytes, which is required for the continuation of the life cycle and aimed at achieving gamete fertilization in the mosquito host. Microgametocytes and macrogametocytes have different morphologies as early as stage III, while stage IV through stage V gametocytes exhibit more pronounced sex dimorphism. In contrast to females, which have a bluish Giemsa stain, a comparatively large nuclear material, and highly intense pigment, males give pink coloration with a larger nucleus, reticular chromatin dispersion, and more pigment. Mature macrogametes (female gametocytes) have a vast endoplasmic reticulum and a large number of ribosomes, which enable rapid and subsequent protein synthesis. Males have chromosomal kinetochores and the microtubule organizing core, which is compatible with the need for fast multiplication of the cell and significant organization of various organelles for the period of gametogenesis [72]. Osmiophilic bodies are present in both macro- and microg ametocytes, but macrogametocytes are substantially more prevalent [71].

From stage IV forward, when gametocytes have an elongated morphology with pointed ends, the differences between male and female gametocytes are most readily apparent morphologically. Female gametocytes have a relatively tiny nucleus, has a nucleolus, and are heavily pigmented. Male gametocytes, which lack a nucleolus and appear as pink cells on Giemsa-stained blood films as opposed to violet females have a larger nucleus and more diffuse pigment [34]. Male-specific marker genes can be found in gametocytes as early as stage I/II; however, utilizing Giemsa-stained blood smears, male and female gametocyte morphological differentiation is not visible until later stages IV and V. In P. falciparum, 206 male and 41 female gametocytes showed differential expression of 247 out of 2110 proteins. Significant numbers of mitochondrial and ribosomal proteins are present in female gametocytes, where they are ready for active protein synthesis at the start of gametogenesis. Since microgamete (male gametocyte) will complete full cycles (three rounds) of DNA copying after initiation, thereby producing eight gametes, that are thin, highly motile, and flagellum-like. These gametes are highly enriched in proteins related to axonemes and flagella, as well as those involved in DNA replication.