Phenotypic screens have partially filled the void in the antimalarial drug market, but as compound libraries eventually become exhausted, new medicines will only come from directed drug development based on a better understanding of fundamental parasite biology.
This review focusses on the unusual cell cycles of Plasmodium , which may present a rich source of novel drug targets as well as a topic of fundamental biological interest. Plasmodium does not grow by conventional binary fission, but rather by several syncytial modes of replication including schizogony and sporogony. Here, we collate what is known about the various cell cycle events and their regulators throughout the Plasmodium life-cycle, highlighting the differences between Plasmodium , model organisms and other apicomplexan parasites and identifying areas where further study is required.
The possibility of DNA replication and the cell cycle as a drug target is also explored. Finally the use of existing tools, emerging technologies, their limitations and future directions to elucidate the peculiarities of the Plasmodium cell cycle are discussed.
It pursues a complex, two-host life-cycle involving both mosquito and human hosts, in which each bottleneck is followed by a replication phase Fig. There are four periods of mitotic DNA synthesis and one period of meiosis during the course of the Plasmodium life-cycle [ 2 ]. The properties of cell division at these replication phases differ fundamentally from conventional models of eukaryotic cell division: rather than binary fission, the parasite opts primarily for schizogony whereby a multinucleate syncytium is formed, prior to budding and cytokinesis [ 3 ].
Equally intriguing is the remarkably rapid process of gamete formation, where male gametocytes undergo three rounds of DNA replication in a matter of minutes, producing eight male gametes [ 4 , 5 ]. Schematic showing the life-cycle of P. Each replicative stage of the life-cycle, together with the approximate fold-replication, is highlighted in purple.
Approximate parasite numbers within each host at each stage are also shown to highlight the severe bottlenecks and massive expansions that occur throughout the life-cycle. Such a complex life-cycle presumably requires sophisticated global and local regulators, involving refined checkpoint and DNA repair mechanisms [ 3 ], yet these are currently only poorly understood.
Along with the unusual spatial and temporal dynamics of DNA replication, cell cycle regulators have been shown to be distinct from human counterparts [ 6 ]. Thus, replication in Plasmodium appears to be an excellent drug target: its mechanisms and regulators are distinct from those of the host organisms, the scale of reproductive output is directly crucial to pathogenicity, and it offers the possibility of interfering with the transmissibility of the parasite.
Furthermore, the parasite possesses two organelles of bacterial origin, the apicoplast and mitochondrion, both of which carry their own genomes and may harbour distinct drug targets in the form of prokaryotic-type replication proteins apicoplast replication was recently well-reviewed [ 7 ], so this article focusses only on nuclear replication.
The standard eukaryotic cell cycle follows a clearly defined series of stages during which the cell grows interphase , replicates its chromosomes S phase and divides M phase , with S phase often being flanked by two gap phases called G1 and G2 Fig.
The process is tightly regulated by an extensive regulatory network that ensures the cell is ready to progress onwards through the cycle after each phase [ 8 , 9 ]. Apicomplexans, including Plasmodium spp. At the heart of these adaptations is schizogony: a syncytial and yet asynchronous form of replication and cell division, which occurs in the mammalian host during hepatic and erythrocytic infections, while a largely analogous process called sporogony occurs in the mosquito vector during oocyst formation Fig.
Schematic of the conventional eukaryotic cell cycle, highlighting the points at which cycle checkpoints operate. For the most virulent human malaria parasite, P. Of these, about two-thirds can successfully invade new erythrocytes, resulting in growth rates of up to fold per hour cycle [ 10 , 11 ].
Finally, the extremely rapid replicative process of male gametogenesis takes just 10—15 minutes Table 1. Thus, there are huge variations of both speed and magnitude between the various replicative stages of the Plasmodium life-cycle.
The Plasmodium cell cycle is best characterised during erythrocytic schizogony, with the start of each cycle being associated with the invasion of a new erythrocyte by a merozoite. During the first 24 hours post-invasion hpi , ring and early-trophozoite parasites possesses a single haploid nucleus in interphase or G1.
The centriolar plaque CP , which is functionally equivalent to the centrosomes in higher eukaryotes, begins to assemble and duplicate 20—24 hpi, marking the shift from growth to replication. Replication of the chromosomes initiates at estimated times of 29 hpi [ 12 ] or 24—26 hpi [ 13 ], overlapping with the semi-conservative duplication of the CP. It was initially proposed that several rounds of continual chromosome replication would then occur, followed by a single co-ordinated mass chromosome segregation, nuclear division and formation of daughter merozoites [ 14 ]; however, an alternative model entails repeated cycles of replication, segregation and nuclear envelope division, based upon the observed segregation patterns of the CP [ 13 ].
In this model the characteristic asynchronicity of schizogony correlates with the inheritance of the CP, with nuclei that inherit the larger mother CP being ready to divide sooner than those nuclei with the daughter CP. It is not known what signal induces a schizont to stop its asynchronous nuclear divisions and undergo a final - and apparently synchronous [ 13 ] - division, followed by budding into membrane-bound daughter cells.
This may also be true for Plasmodium , although schizogony is numerically more complex than endodyogeny in T. Overall, significant gaps remain in our understanding of the cell biology underlying schizogony, and even less is known about the other phases of Plasmodium replication Fig. Many early-diverging eukaryotes, such as Trypanosoma and Oxytricha , organise their genomes in extremely unusual ways [ 19 , 20 ]; by comparison, the basic genome structure of Plasmodium is quite conventional.
There are 14 linear chromosomes with telomeres and centromeres, plus two small organellar genomes in the mitochondrion and apicoplast [ 21 ]. This conventional genome is replicated, however, within the framework of fundamentally unusual cell biology. Although much remains to be studied, some of the proteins and parameters involved in this process have now begun to be elucidated. Plasmodium encodes the basic replicative machinery that is found in all eukaryotes, including DNA polymerases [ 21 , 22 ], proliferating cell nuclear antigen PCNA [ 23 , 24 ] and minichromosome maintenance proteins MCMs [ 25 , 26 ].
The remaining members of the complex are either absent or lack sufficient homology with characterised members to allow clear identification, although in P.
Cdc6 is required for the recruitment of Cdt1 and the loading of MCM proteins to date only a putative Cdt1-like gene has been identified in Plasmodium. Sequence analysis has failed to identify a clear homologue of Cdc45 in Plasmodium , while the members of the GINS complex have been putatively identified, based on low sequence homology, but it remains to be determined whether they are functional.
It recognises a conserved consensus sequence in the yeast Saccharomyces cerevisiae , but in other eukaryotes there is no consensus sequence and the preferred composition of DNA bound by ORC varies from organism to organism [ 29 ]. Putative ORC-binding sequences in P. The rate of replication was not constant, but decreased as the cells neared completion of schizogony, coinciding with a reduction in the mean distance between individual origins.
Interestingly, this is the opposite of the pattern seen in human cells, where replication speeds up and origins become more widely spaced as S phase proceeds. Development of techniques to examine the replication of the Plasmodium genome. Origins in S. Following reversible DNA-protein cross linking, the genome is fragmented and the proteins of interest are purified along with the associated DNA fragments, which are then sequenced.
This may include origins that would never be activated, and may miss those where the protein complex has dissociated from the chromosome. Parasites expressing viral thymidine kinase can incorporate the synthetic nucleosides IdU red and CldU green which can be visualised in individual nuclei or on combed DNA fibres, allowing the calculation of inter-origin distances and replication rates. During erythrocytic schizogony the first replication and possible nuclear division requires 4—6 hours [ 13 ].
This, however, is not the maximal rate of replication for the parasite. After ingestion by a mosquito, male gametocytes undergo a 3-fold replication of their genome in less than 15 minutes, resulting in the production of 8 motile microgametes with 1N genome content [ 4 , 5 ].
Female gametocytes also mature and exit their host cells upon entering a mosquito but no replication or cell division occurs. In the model rodent malaria species P. Such extreme speed is unprecedented in eukaryotic gametogenesis, and may reflect strong pressure to complete the sexual cycle and exit the midgut before the parasite cells are digested along with the blood meal.
If the replication speed remains the same as it is in erythrocytic schizogony, then almost all of the suggested ORC-binding sites [ 30 ] must be used as origins simultaneously. Precedents do exist for such extremely flexible origin usage: in the earliest replications of Xenopus embryos, origins occur every 5—15 kb, spacing out only after the mid-blastula transition [ 33 ]. Plasmodium genome replication may be under similarly flexible control, although nothing is yet known about how this might be differentially enforced in gametogenesis, sporogony, hepatic and erythrocytic schizogony.
As described above, Plasmodium undergoes multiple unconventional cell cycles, in a variety of host cell types and for varying durations. Although the genomic revolution for Plasmodium has permitted some investigation of these regulators, our understanding at present is patchy and incomplete. In eukaryotic cells, cell cycle progression is governed by cyclins and cyclin-dependent protein kinases CDKs , along with other proteins such the anaphase promoting complex APC , which promotes waves of cyclin degradation.
The interplay between these regulatory and catalytic components and their timely upregulation, inhibition and degradation prompts sequential progression through G1, S, G2 and M phases [ 34 ] Fig. The peculiarities of Plasmodium schizogony begin with the lack of a G2 phase as the syncytial nuclei appear to alternate asynchronously between S and M phases prior to the orchestrated event of cytokinesis [ 35 ] Fig. This raises questions about whether control of replicative cycles through diffusible cytoplasmic factors is feasible [ 2 , 12 ].
Illustration of cell cycle phases in Plasmodium erythrocytic schizogony a and Plasmodium male gametogenesis b. The predicted involvement of cyclins, CDKs and other kinases is shown at each phase.
Placement of such components is only loosely chronological since most details are unknown. Crks or CDKs predicted to be involved in transcriptional regulation are transparent without a white background.
Interactions identified in vitro between cyclins and CDKs are indicated by a dashed orange arrow. Table 2 identifies all sources used to construct the figure. The cell cycles at sporogony and hepatic schizogony are omitted due to the lack of information about these stages. None of the cyclins are homologs of canonical cell-cycle cyclins e. Although PK5 is the putative homologue of mammalian CDK1, Plasmodium encodes no cognate cyclins for such an enzyme and the activator for PK5 remains unknown: in vitro , it is unusually promiscuous and can be activated by all three Plasmodium cyclins as well as mammalian p25, cyclin H and RINGO [ 37 , 44 , 45 ].
The partnership with Cyc1 is questionable because recent immunoprecipitation studies failed to identify it [ 46 ]. Mrk1 is not apparently a CDK-activating kinase in Plasmodium and although it can interact in vitro with the replication factor complex PfRFC-5 and PfMCM6 [ 40 ], it actually appears to be crucial for cytokinesis rather than replication, as indeed is Cyc1 [ 46 ], with Mrk1 acting in a complex with Cyc1 and MAT1 [ 46 ].
Crk5 can be activated in vitro by Cyc1 and Cyc4 but its in vivo partner is again unknown; it is involved in, but not essential for, erythrocytic schizogony because its absence results in viable parasites with fewer merozoites per schizont [ 43 ].
PK6 is proposed to be involved in the onset of S phase in erythrocytic stages but in vivo characterisation is lacking, and recombinant PK6 is cyclin-independent in vitro [ 47 ]. The remaining CDKs, Crk-1 and Crk-3, are predicted to have roles in transcriptional regulation and thus in cell growth and proliferation [ 42 , 48 ].
Another - perhaps more interesting - group of regulators are specific to the unusual cell cycle modes of apicomplexans and there is considerable interest in the plant-like calcium-dependent protein kinases CDPKs as possible parasite-specific drug targets, with CDPK4 playing multiple roles in male gametogenesis [ 28 ] and CDPK7, in erythrocytic schizogony [ 53 ].
Another Plasmodium -specific kinase, PfCRK4, was recently identified as essential for DNA replication in erythrocytic schizogony, although the pathway in which it acts remains to be elucidated [ 12 ].
In addition to the cyclin-CDK regulatory network, there are also defined checkpoints in yeast and mammalian systems that control cell cycle advancement. These serve as quality control for cell growth G1 checkpoint , successful DNA replication or DNA damage S and G2 checkpoints and chromosome attachment to the spindle M checkpoint [ 54 ].
Checkpoints are particularly important for avoiding re-replication and preventing the propagation of incompletely replicated or damaged daughter genomes. The existence of cell cycle checkpoints in Plasmodium remains, in general, uncertain, and genes encoding key checkpoint proteins such as Rb, p53, ATM and ATR have not been identified. There is, however, some evidence of a G1 checkpoint in the related parasite T.
What induces these states in the absence of a conventional G1 checkpoint pathway is unclear. DNA repair machinery is largely conserved in the parasite genome, as described below, and parasites respond to DNA damage by upregulating repair machinery and altering chromosome structure [ 60 ].
However, there is no apparent G2, offering no opportunity for a G2 checkpoint [ 13 , 35 ] and the feasibility of intra-S and M checkpoints is challenged by the striking variation in the speed of genome replication at different life-cycle stages, particularly the unprecedented rate in male gametocytes [ 61 ], sharply contrasting with a more conventional rate during erythrocytic schizogony [ 13 , 31 ].
Checkpoint regulation may be temporally possible during erythrocytic schizogony - and perhaps also oocyst sporogony and hepatic schizogony - but not gametogenesis.
Spatially, schizogony also poses challenges to checkpoint control. Although chromosomes do appear to align with the hemispindle, which is anchored to a CP, they remain uncondensed: it is thought that the centromeres remain constantly attached to CPs and that this may help to separate the uncondensed chromosomes accurately [ 3 ].
Finally, the syncytial nature of Plasmodium replication raises questions about diffusible checkpoint factors and the how the replication of individual genomes could be stalled within a shared cytoplasm [ 2 ].
Variations in cell cycle speed also raise questions about replicative fidelity and tolerance of karyotypic variation. Under drug pressure, P. This permits fine-tuning of amplicon numbers, relevant to drug pressure, while avoiding genome damage and any deleterious mutations in off-target loci [ 63 ].
Two recent studies have suggested that the mitotic mutation rate does not vary between P. In this regard, P. The extremely fast replication of male gametes raises a particular conundrum in terms of checkpoints: does this phase require especially stringent regulation, or conversely, more relaxed control to favour speed over fidelity?
The observation that some male gametes are produced with apparently partial or absent nuclei unpublished observations and the fact that male-expressed genes display fast rates of evolution [ 71 ] may suggest the latter. Proteins involved in DNA replication and mitosis are simultaneously phosphorylated within the first 20 seconds of gametocyte activation, contrary to the traditional view of sequential progression through the cell cycle, and this may facilitate the rapidity of gametogenesis [ 32 ].
Indeed, the relatively limited repertoire of cell cycle kinases in Plasmodium may also imply that some have dual functions: CDPK4 has been implicated in assembly of the pre-replicative complex, mitotic spindle formation, cytokinesis and axoneme motility [ 28 , 32 , 72 ]. Regardless of cell cycle speed, the parasite is clearly able to promote genomic diversity during mitosis, as well as more conventionally at meiosis. It seems unlikely that the intricacy and precision of the Plasmodium cell cycles would proceed unchecked, but evidence is currently lacking for clearly defined checkpoints and there may be great flexibility in which checkpoints are enforced during different types of replication.
DNA damage can originate from a range of sources, the most common in Plasmodium being reactive oxygen species generated by metabolism, free radicals, which are often produced after uptake of antimalarial drugs such as chloroquine or artemisinin, and errors made during DNA replication.
Damage may affect individual bases or may lead to the generation of potentially deadly double strand breaks DSBs. The mutational spectrum observed in P. This can promote the formation of pseudopolyploid loci, as described above, but the core genome nevertheless remains intact, due to the presence of an effective DNA repair system including most - although notably not all - of the pathways commonly found in model eukaryotes.
Orthologs of the majority of genes involved in the NER pathway have been identified bioinformatically, with the exception of p62 and XPC [ 73 ]. Similarly, the majority of the MMR pathway is present but there are notable differences from other eukaryotes, with RecJ exonucleases appearing to be absent while a UvrD helicase homolog, found in E.
The majority of eukaryotes rely upon two major pathways for the repair of double-strand breaks, homologous recombination HR and non-homologous end joining NHEJ. The Plasmodium genome encodes a functional HR pathway but the core genes of the NHEJ pathway appear to be absent across the genus [ 21 , 77 ], supported by the inability to detect NHEJ products in vitro after the experimental generation of DSBs [ 78 , 79 ].
HR, indeed, appears to be essential for the completion of the parasite life-cycle because the knockout of a zinc finger protein, Pb Zfp, in P. During all haploid growth phases the parasite must therefore rely upon alternative end joining pathways such as microhomology-mediated end joining MMEJ to repair DSBs within the core genome, because no repair template exists to allow HR [ 84 ].
Bioinformatic comparisons with the S. Notably, this restriction does not apply to multigene families, such as the var family of key virulence genes in P. This leads to important diversification of these gene families during mitotic growth [ 68 ] as well as during meiosis , generating new antigenic variants that can facilitate immune evasion during chronic human infections.
Var gene recombination does not require substantial stretches of high sequence homology and the genes do not necessarily recombine with their closest homologues [ 68 ]; the physical clustering of var genes at the nuclear periphery may favour sequence pairing even in the absence of extensive homology.
Historically, DNA replication has been an excellent drug target in malaria parasites, as demonstrated by Fansidar: an anti-folate drug combination which proved vital after the emergence of chloroquine resistance in the late s [ 88 ].
Fansidar is a synergistic combination of two drugs that block the pathway to production of reduced folate cofactors essential for nucleotide production and DNA synthesis , but resistance to the combination arose fairly rapidly [ 89 , 90 ].
However, directly targeting the regulatory machinery of the parasite, such as cell-cycle checkpoint control, or eliciting DNA damage as a route to parasite killing, may provide a greater hurdle to resistance development. Indeed, DNA damage, together with protein damage, is thought to be a mode of action for the frontline antimalarial drug artemisinin, mediated through free radicals [ 91 ].
For Plasmodium , understanding the cell cycle arrest phenotype takes on new urgency because it is considered a key mechanism of artemisinin resistance [ 94 ]. This resistance is not yet fully understood in molecular terms, but it correlates with mutations in the Kelch protein, which in turn correlate with elevated levels of the phosphoinositidekinase enzyme PfPI3K [ 95 ]. PfPI3K, a lipid kinase, is distantly related to protein kinases that are key checkpoint proteins in most eukaryotes but are missing in Plasmodium - an intriguing similarity that is currently under investigation in our laboratory.
In rapidly dividing human cells with a hour cell cycle, the G 1 phase lasts approximately nine hours, the S phase lasts 10 hours, the G 2 phase lasts about four and one-half hours, and the M phase lasts approximately one-half hour. In early embryos of fruit flies, the cell cycle is completed in about eight minutes. The timing of events in the cell cycle is controlled by mechanisms that are both internal and external to the cell. Both the initiation and inhibition of cell division are triggered by events external to the cell when it is about to begin the replication process.
An event may be as simple as the death of a nearby cell or as sweeping as the release of growth-promoting hormones, such as human growth hormone HGH. Crowding of cells can also inhibit cell division. Another factor that can initiate cell division is the size of the cell; as a cell grows, it becomes inefficient due to its decreasing surface-to-volume ratio.
The solution to this problem is to divide. Whatever the source of the message, the cell receives the signal, and a series of events within the cell allows it to proceed into mitosis. Moving forward from this initiation point, every parameter required during each cell cycle phase must be met or the cycle cannot progress. It is essential that the daughter cells produced be exact duplicates of the parent cell.
Mistakes in the duplication or distribution of the chromosomes lead to mutations that may be passed forward to every new cell produced from an abnormal cell. To prevent a compromised cell from continuing to divide, there are internal control mechanisms that operate at three main cell cycle checkpoints.
A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable. Each step of the cell cycle is monitored by internal controls called checkpoints.
Positive regulator molecules allow the cell cycle to advance to the next stage. Negative regulator molecules monitor cellular conditions and can halt the cycle until specific requirements are met. Cancer comprises many different diseases caused by a common mechanism: uncontrolled cell growth. Despite the redundancy and overlapping levels of cell cycle control, errors do occur. One of the critical processes monitored by the cell cycle checkpoint surveillance mechanism is the proper replication of DNA during the S phase.
Even when all of the cell cycle controls are fully functional, a small percentage of replication errors mutations will be passed on to the daughter cells. It is thus evident that the recent advances made in understanding the cell cycle machinery and the DNA damage checkpoint response have identified a wealth of new targets for the development of new antineoplastic agents, or of response modifiers for existing ones.
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