DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Jennifer McDonald; Catherine J Merrick

doi:10.1371/journal.ppat.1010595

. 2022 Jun 22;18(6):e1010595. doi: 10.1371/journal.ppat.1010595

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Jennifer McDonald ¹, Catherine J Merrick ^1,^*

Editor: Michael J Blackman²

PMCID: PMC9255763 PMID: 35731838

Abstract

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This involves the asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite reproduction is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear multiplications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active DNA replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their DNA replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

Author summary

Malaria parasites are unusual, early-diverging single-celled organisms. One of their atypical features is their mode of producing new cells. Most cells replicate their genome, segregate the copies into two nuclei and then split the whole cell in two: a process called binary fission. Malaria parasites, by contrast, multiply primarily by schizogony. Each cell replicates its genome several times over, asynchronously, generating many nuclei within the same cytoplasm, before splitting into many new daughter cells in a single mass event. All malaria species do this, but there are stark differences between species in how long schizogony takes and how many progeny each cell can produce. Understanding this is important because the rate of parasite reproduction is fundamentally important to malarial disease: humans who have a high burden of parasites in their blood are most likely to suffer severe malaria. Here we compare the process of schizogony in two different species, by developing cell lines in which we can follow de novo DNA replication at high spatial and temporal resolution, at both whole-cell and single-molecule levels. We establish the dynamics of schizogony, highlighting similarities and differences between two species and setting parameters for future studies of interventions that could interfere with parasite reproduction.

Introduction

The genus of malaria parasites Plasmodium lies in an early-diverging protozoan lineage with many unusual features. One such feature is the type of cell cycle that this parasite pursues [1]. Plasmodium does not divide by binary fission–the well-studied process that occurs in almost all model systems from yeasts to human cells. Instead, new cells are produced primarily by schizogony, a unique process involving asynchronous DNA replication in multiple nuclei within the same cytoplasm, prior to a mass cytokinesis event producing many–and not necessarily 2ⁿ –daughter cells called merozoites [2].

Schizogony occurs first in hepatocytes and then repeatedly in erythrocytes, causing all the pathology of malaria. The process is clinically important because the rate of growth in erythrocytes determines parasitaemia, which often correlates with disease severity [3, 4]. Parasitaemia in human malaria can vary greatly, partly because the human-infective Plasmodium species (P. falciparum, P. vivax, P. malariae, P. vivax, P. knowlesi and P. ovale wallikeri & curtsii) differ in their preferences for human erythrocytes. For example, P. vivax is restricted to invading scarce reticulocytes, P. malariae primarily invades older erythrocytes, and P. falciparum invades cells of all ages and can therefore reach high parasitaemias as extreme as 40% [5].

There are also other inter-species differences in the process of schizogony. Its duration varies from ~72 hours in P. malariae to ~24 hours in P. knowlesi and the number of merozoites produced per schizont varies from as many as ~30 in P. falciparum to ~15 in P. knowlesi. Exactly what limits merozoite numbers to a characteristic range for each species is unknown, but it is evidently not the fundamental size of the human erythrocyte, nor the duration of schizogony. Moving from the cell-biological to the molecular level, the composition of Plasmodium genomes also varies considerably between species. P. falciparum has an extremely biased genome at ~81% A/T [6] whereas P. vivax and P. knowlesi have A/T contents of only 60 and 61% [7, 8]. The reasons for these differences, and their potential influence upon the speed and fidelity of DNA replication, are unknown.

To better understand the dynamics of schizogony, we have compared the process in the only two human-infective species that are amenable to culture, P. falciparum and P. knowlesi. These differ in parameters such as genome content, cell cycle period and merozoite number. Little has been published on this subject in P. knowlesi but we have previously explored DNA replication dynamics in P. falciparum [9]. To do this we created a parasite line that allows de novo DNA replication to be followed at high resolution via the incorporation of pulse-labels of modified nucleotides [10]. Thus, we defined DNA replication dynamics at the single-molecule level [9] and, together with prior work at the cellular level [2, 11], this provided an overall picture of P. falciparum erythrocytic schizogony. Here, we have added considerable detail to that picture and used the same DNA-replication-labelling technology to compare P. knowlesi–a species that completes schizogony twice as fast, makes fewer merozoites, and has a more balanced genome composition.

To summarise what is already known about P. falciparum schizogony, the first round of DNA replication begins more than halfway through the cell cycle (which is theoretically 48h, actually ~42-50h in different strains and culture systems [2, 12]). Four or five rounds of nuclear multiplication occur and there is karyokinesis at each round [2, 11], without cytokinesis or conventional G1/G2 phases [2, 11, 13]. Duplication of the centriolar plaque (the Plasmodium centriole-equivalent) has been proposed to initiate each round of DNA replication [2]. Asynchrony, which is clearly seen in the early rounds, has been linked to partitioning of centriolar plaques, with the nucleus that receives the more mature plaque commencing its next DNA replication first [2]. Without the benefit of DNA replication labelling, prior work could not define the later replicative rounds precisely, as nuclei become very crowded inside the erythrocyte, but the final round was suggested to be synchronous [12, 14]. The degree of synchrony has since been disputed, most recently via high-resolution electron-microscopy data [2, 15]. Indeed, it is unclear how a final synchronous round would be coordinated if centriolar plaques of differing maturity do drive the rate of DNA replication. Other unanswered questions include: is there a limit on the number of nuclei that can replicate DNA at once, perhaps via a limiting factor, and how is this overcome if the final round is synchronous? Do all successive rounds of nuclear multiplication take the same time? Are there consistent gaps between replicative rounds (G1 or G2 equivalents), or do some nuclei ‘stall’–adding to asynchrony and suggesting a DNA replication checkpoint?

At the single-molecule level, our recent work defined the average speed of replication forks and spacing of replication origins in P. falciparum schizogony at 1.2kb/min and 65kb between origins–broadly similar to parameters in other eukaryotic cells. These averages changed by ~30% over the course of schizogony, with the fastest fork movement and most widely-spaced origins occurring early on [9]. The opposite pattern occurs in human cells, where DNA replication speed is usually limited by cellular nucleotide pools and becomes fastest towards the end of S-phase as nucleotide production peaks [16]. By contrast, in P. falciparum, we speculated that the nucleotide pool could become increasingly limiting as more nuclei replicate their DNA at once, and/or that DNA condensation or accumulated DNA damage might limit the pace of replication [9]. In P. knowlesi, fewer nuclei are packed into each cell and the genome composition is different from that of P. falciparum, so the parameters of DNA replication could conceivably be very different. Accordingly, we have now measured these parameters at both the cellular and single-molecule levels in both species.

Results

Timecourse experiments across S-phase in P. falciparum and P. knowlesi

To follow DNA replication dynamics throughout schizogony in both P. falciparum and P. knowlesi, we first transfected P. knowlesi with a thymidine-kinase-expressing plasmid, as previously performed in P. falciparum [10]. We confirmed that this permitted the labelling of DNA replication with modified nucleotides (bromo/chloro/iodo/ethyl-deoxyuridine (BrdU/CldU/IdU/EdU)), and that parasite behaviour in terms of fitness and cell-cycle timing was not overtly disturbed (S1 Fig).

We then conducted time-course experiments with highly synchronised cultures of both lines. Synchronisation was achieved by purifying late-stage schizonts, allowing 1h of reinvasion, then purifying the resultant ring-stages to yield a 1h window around the point of invasion (termed ‘0 hpi’). Aliquots of parasites were pulse-labelled with EdU for 30 minutes every hour throughout the period of DNA replication, then immediately fixed and prepared for microscopy. All the nuclear masses in each cell were detected with a DNA stain, and all those that were actively replicating were detected via EdU click chemistry. (A nuclear mass refers to a distinguishable mass of DNA that may or may not be a discrete nucleus–i.e. fully enveloped in a nuclear membrane–since we were unable to stain this membrane.) Centrin foci were also detected by immunofluorescence [2], to quantify the centriolar plaques associated with each nuclear mass. Fig 1A shows a schematic of the experimental setup. In both species, additional experiments were then conducted, using two distinguishable pulses of modified nucleotides at increasing time intervals (Fig 1B and 1C). This allowed the detection not only of nuclei actively replicating their DNA at time x, but also of nuclei that had been replicating at time x-y. Thus, we could determine the length of each replicative round and of the gaps between rounds.

Fig 1 — A: Schematics of timecourses using single EdU pulse labels. B: Schematic of experiments using sequential pulses of two nucleotides (EdU and BrdU) to determine the length of each replicative round. C: Schematics of timecourses using 2-nucleotide (EdU and BrdU) interval pulse labels.

The length of S-phase is proportional to the overall length of erythrocytic schizogony

To measure the overall parameters of S-phase, we first counted all the nuclear masses, and all those that were actively replicating DNA, in 100 cells at each timepoint listed in Fig 1A (S2 Fig). P. falciparum schizonts produced 5–28 nuclei while P. knowlesi produced 3–16 (Fig 2A and 2B). Median numbers of nuclei at the most mature timepoint were 11 for P. falciparum and 6 for P. knowlesi, but the numbers produced in both species were highly variable: amongst populations of late-stage schizonts, maximum numbers of merozoites were 28 and 16, but rare cells were still visible with only 4 or 2 nuclei respectively (Fig 2A and 2B). This could be because a small minority of cells were very delayed in starting S-phase, or because a minority underwent fewer/slower rounds of nuclear multiplication. Favouring the latter explanation, the onset of S-phase for the majority of cells was quite synchronous (Fig 2C and 2D), particularly in P. knowlesi, where ~60% of cells commenced DNA replication within a single hour at 22-23hpi. Median numbers of nuclear masses counted at the latest timepoints were markedly lower than in previous studies that counted only mature schizonts, arrested or visually selected at the point of egress [12, 17]. This is because we included all cells, counted agnostically at each timepoint, rather than only segmented schizonts, and therefore captured the full diversity of the population, including cells that may have experienced S-phase delays and/or fewer rounds of nuclear multiplication.

Fig 2 — A, B: Scatter graphs of nuclear numbers at each hour in P. *falciparum* (A) or P. *knowlesi* (B), n = 100 cells, medians are shown in red. Biological replicate experiments are shown in S3 Fig. C, D: Percentage of 100 cells showing some EdU labelling at each hour in P. *falciparum* (C) and P. *knowlesi* (D). E, F: Percentage of replicating nuclear masses per cell at each timepoint in P. *falciparum* (E) and P. *knowlesi* (F), n = 100, medians are shown in red. G, H: Percentage of replicating nuclear masses per cell, re-plotted by number of such masses, in P. *falciparum* (G) and P. *knowlesi* (H), n = 100, medians are shown in red.

The overall length of S-phase was estimated as the time between the first appearance of cells with actively replicating DNA and the time when the number of individual nuclear masses stopped increasing. In P. falciparum, cells with replicating DNA began to appear at 31-32hpi and cells with more than 1 nuclear mass (having completed the first S-phase) began to appear at 32hpi (Figs 2A and 2C, and S3). Numbers of nuclear masses stopped increasing at 46-47hpi, making 15 hours the total S-phase period across the population (importantly, this may not apply to every cell within that population, and could also be slightly influenced by strain-to-strain variation in strains other than 3D7). In P. knowlesi, DNA replication started at 22hpi, 2n cells began to appear at 23hpi, and numbers of nuclear masses stopped increasing at 31-32hpi (coinciding with a sharp drop in EdU labelling from 31hpi), i.e. an S-phase period of only 9h (Figs 2B and 2D, and S3). Notably, this P. knowlesi timecourse was followed until almost all the schizonts had burst at 33hpi, confirming a lag of just 2h between the cessation of DNA replication and the completion of reinvasion. In P. falciparum, this first timecourse was not followed through to the end of reinvasion but a replicate experiment (S3 Fig), as well as the subsequent EdU/BrdU labelled timecourse would confirm that P. falciparum showed substantial reinvasion by 48hpi (S3 Fig), and so has a similar window of ~2h between the end of S-phase and reinvasion. Within this window, daughter merozoites must be fully assembled and cytokinesis must occur.

These data showed that the proportion of the cell cycle occupied by S-phase was very similar in both species, at ~30% (15 of 48h, or 9 of 31h) and that the lag period between nuclear multiplication and reinvasion was similarly short in both species. All parameters shown in Fig 2 were broadly reproducible in separate timecourses conducted as full biological replicates in different blood batches on different days (S3 Fig). Variation in batches of host erythrocytes can influence Plasmodium cell cycles, as documented in previous work [12, 17]. In our replicates, S-phase in P. falciparum proceeded with similar timing to the previous timecourse, between ~32-47hpi, but a slightly higher peak in median numbers of nuclear masses was counted at 47hpi. The replicate timecourse in P. knowlesi produced similar numbers of nuclear masses but was set forward by several hours (and likewise in the subsequent EdU/BrdU labelled timecourse). In our hands, the cycle time of P. knowlesi is particularly sensitive to different batches of human erythrocytes: ~28h is common in culture and the initial timecourse shown in Fig 2 was therefore unusually long, possibly due to the age of the erythrocytes in that blood batch.

There is no restriction on the number of nuclei that can replicate at once

DNA replication in schizogony is clearly a stochastic, asynchronous process and different nuclei within the same cell can simultaneously experience either DNA replication or resting ‘gap’ phases [2, 11]. We therefore examined whether there is a limit on the number of nuclei that can replicate simultaneously. If, for example, the pool of nucleotides or replication proteins is limiting, we should observe a limit on the number of nuclei per cell that can replicate their DNA within a 30-minute period. In fact, throughout schizogony this was not true. In the earliest stages of S-phase, the percentage of labelled nuclear masses was either 50 or 100%, since cells had only 1 or 2 nuclei. Subsequently, however, cells appeared with anything from 20% to 100% of their distinguishable nuclear masses actively replicating DNA. The median percentage at most timepoints was 60–70% in P. falciparum and 50–60% in P. knowlesi (Fig 2E and 2F).

To obtain a clearer view of whether the number of nuclei replicating their DNA at once depended on the number of nuclei existing in a schizont, we re-plotted the data according to number of existing nuclear masses rather than hpi (Fig 2G and 2H). This confirmed the conclusion that a schizont with any number of nuclei could replicate DNA in any proportion of those nuclei within a 30-minute window. Notably, in P. knowlesi DNA replication was rarely observed after the stage of 8–9 nuclear masses (Fig 2H), consistent with this species reaching a maximum of 16 nuclei, and with the final round of DNA replication being quite, but not entirely, synchronous. By contrast, numbers of nuclear masses in P. falciparum schizonts were highly variable and cells with 15 or more nuclei were therefore binned together (Fig 2G), but there was no single number of nuclei (even above 15) at which DNA replication ceased, indicating greater variability and asynchrony in P. falciparum.

Patterns of DNA replication within nuclei vary from early to late schizogony

In both P. falciparum and P. knowlesi, it was clear that the sub-nuclear distribution of actively-replicating DNA during the 30-minute pulse-label varied in different nuclei (Fig 3B). The commonest pattern was a completely labelled nuclear mass with signal detected throughout the DAPI-stained area, showing that DNA in all areas had replicated in the previous 30 minutes. Amongst these, and particularly notable in P. falciparum, was a subset with homogenous but fainter staining, suggesting that DNA had replicated much less rapidly than in the common pattern: this appeared mostly in late-stage cells completing their presumptive final round of nuclear multiplication (Fig 3C, see P. falciparum at 44-45hpi, P. knowlesi at 30hpi, and quantification in Fig 3D and 3E). Other patterns included nuclear masses with only a partial area showing DNA replication, and nuclear masses with discrete foci of DNA replication: these appeared at low frequencies throughout both timecourses (Fig 3D).

To quantify these observations, we categorised DNA replication patterns in the nuclei of 100 cells at each timepoint (Fig 3D). We also quantified the amount of de novo DNA replication occurring within the pulse-label via the density of EdU staining per nuclear mass (Fig 3E), confirming the late-stage dominance of the ‘homogenous faint’ DNA replication pattern (individual patterns are shown as separate graphs in S4 Fig). Marked differences appeared between P. falciparum and P. knowlesi. Firstly, nuclear masses with the ‘partial’ DNA replication patterns made up 20–30% of the total in P. falciparum at all timepoints, but fewer than 10% of the total in P. knowlesi at all times until the final timepoint (Fig 3D). Secondly, the majority of nuclear masses in P. falciparum showed the faint staining pattern at 44-47hpi, whereas in P. knowlesi this pattern increased slightly but never became dominant, appearing in only ~20% of nuclear masses (Fig 3D and 3E).

Centriolar plaques are dynamic throughout schizogony

Several previous studies have highlighted the importance of the centrosome equivalent–the centriolar plaque–in controlling nuclear multiplication [2, 18] (S5A Fig). We therefore quantified whether or not nuclei in each phase of DNA replication were associated with detectable centriolar plaques, using foci of centrin as a marker. Importantly, these experiments relied upon a commercial anti-centrin antibody, the sensitivity of which was called into question in recent work (published after this study was conducted) [18]. Using this marker, centrin foci were not always detectable (S5B and S2 Figs): overall, ~50% of nuclear masses had detectable centrin foci, varying from <30% in the earliest and latest timepoints to >60% at the midpoint of schizogony (37-42hpi in P. falciparum and 25-28hpi in P. knowlesi (S5C Fig)). Only a minority of nuclear masses, ~25% at most timepoints, showed centrin foci as well as active DNA replication, while the majority showed either DNA replication with no centrin, or centrin with no DNA replication (S5D Fig).

The data were broken down into distinct types of centrin labelling: a single focus, two separate but adjacent foci, and two foci on opposite sides of the nuclear mass (S2 Fig). These are thought to represent sequential events in preparation for karyokinesis (S5A Fig). Across 100 cells, in both P. falciparum and P. knowlesi, duplicated centrin foci were relatively rare, particularly in the opposite configuration, showing that this phase is very brief (S5C Fig). About half of all nuclear masses with two foci showed DNA replication within the past 30 minutes, while the other half did not (S5D Fig). Finally, nuclear masses with two centrin foci were never seen past the stage of 8–9 such masses in P. knowlesi (S5E and S5F Fig). This division was less sharp in P. falciparum, but the appearance of centrin foci did drop off in cells with more than ~10 nuclear masses. In both species, it therefore appears than centriolar plaques are disassembled after the final round of DNA replication and karyokinesis. Beyond this, it was not possible to discern a clear relationship between the pattern of intranuclear DNA replication (full, partial, discrete foci, etc.) and the pattern of centrin foci (S5E and S5F Fig).

Replicative rounds become faster across the course of schizogony

The foregoing experiments could not measure the time taken by each round of nuclear multiplication, because they provided only snapshots at successive timepoints. Therefore, we elaborated the protocol to use two successive, distinguishable pulse-labels (Fig 4A). Cultures were labelled with EdU for 30 minutes (as before), then with BrdU at 15-minute intervals up to 120 minutes in total. In a 1-n trophozoite, a single nucleus bearing both labels (Fig 4B) must have been replicating DNA from at least the end of the first pulse to the start of the second, thus measuring the potential length of S-phase. Accordingly, as shown in Fig 4A, when 30 minutes of EdU label was directly followed by 15 minutes of BrdU, S-phase could be ≥45 minutes, but minimally ~10 minutes (requiring 5 active minutes in each pulse for replicating DNA to be detected with confidence (S6 Fig)). Similarly, with a gap of 75 minutes separating the labels, S-phase must be at least 85 minutes. Fig 4C quantifies this experiment for P. falciparum, showing that at least 70% of nuclear multiplications in 1-n cells took 40–75 minutes, but only ~15% took 55–90 or 70–105, and almost none took >85 minutes (Fig 4C). (Notably, at the 45-minute interval, 35% of nuclei had completed DNA replication within the first pulse but did not label at all with the second, i.e. they paused for at least an hour before karyokinesis. This percentage then dropped, suggesting that very few nuclei paused for >75 minutes before dividing and starting a second round.)

In multinucleate cells, the situation is more complex because a nuclear mass could potentially have completed its DNA replication during the first label, divided in the intervening period, and then picked up the second label in a subsequent replicative round. We assumed that if most rounds of genome replication took 40–75 minutes, this would be unlikely when the interval between labels was ≤30 minutes, and therefore counted such double-labelled nuclear masses as being in the same replicative round. As ploidy increased there was a clear trend towards fewer nuclear masses being double-labelled at increasing time intervals (Fig 4C, compare 2 nuclei with <6 nuclei). Therefore, rounds became progressively faster as ploidy increased.

In P. knowlesi (Fig 4D), the first replicative round took markedly longer, with ~40% of nuclei taking 55–90 minutes, compared to only 15% of the equivalent nuclei in P. falciparum. In fact, ~15% of nuclei took as long as 85–120 minutes (Fig 4E). This marked difference was replicated in separate cultures of P. falciparum and P. knowlesi (S7 Fig). After this, however, subsequent replicative rounds seemed to speed up more than they did in P. falciparum. In P. knowlesi with >6 nuclear masses, only ~25% of those replicated for ≥25 minutes (~25% of nuclear masses showed EdU+BrdU after the 15min time interval), whereas in P. falciparum with >6 nuclear masses, ~40% were still taking at least 25 minutes to complete a replicative round. In fact, in P. knowlesi schizonts with >6 nuclear masses, some of them probably entered the next round within 40 minutes (see Fig 4E, 30min interval).

Gaps between replicative rounds become longer across the course of schizogony

Finally, the interval-labelling protocol shown in Fig 4A was extended over longer periods, with intervals of up to 10h (Fig 5A). This was designed to assess the gaps between rounds of nuclear multiplication (and also to serve as a further replicate, confirming the reproducibility of the initial datasets). Examples of the resultant labelling patterns are shown in Fig 5B. These were quantified in terms of the percentage of nuclear masses labelled with either one or both nucleotides across 50 cells (Fig 5C).

The data indicated that a) gap phases are relatively long, b) they are longer in P. falciparum than P. knowlesi, and c) they become longer in both species over the course of S-phase. In P. falciparum, when sequential labels were separated by 2h (41–43 and 46-48hpi) only ~20% of nuclear masses were double-labelled, showing that relatively few nuclei commenced a second round within 2h (S8 Fig). With 3h intervals, a greater proportion of nuclei re-entered S-phase: ~50% of nuclear masses became double-labelled between 35-38hpi, but this diminished to ~40% and 20% at 38–41 and 41-44hpi (Fig 5C). Therefore, gaps increased over the course of S-phase (and/or progressively more nuclei did not re-enter DNA replication at all), even as the time spent by each nucleus actively replicating its DNA became shorter as schizogony progressed (Fig 4). By contrast, in P. knowlesi, gap phases were generally shorter because markedly more nuclear masses became double-labelled within 2h: ~50% of all nuclear masses in early stages, diminishing again to ~35% and 20% at the final 2h intervals (Fig 5E).

Individual nuclei commonly arrest for long periods during schizogony

To address the question of heterogeneity in gaps–i.e. whether nuclei can arrest for long periods, or even permanently, while others in the same cell continue to replicate their DNA, we labelled cells for 30 minutes with EdU, followed by a continuous 5h BrdU label. Nuclei showing EdU but no BrdU must have arrested for at least 5h after a period of active DNA replication: markedly longer than the common gap period of 2-3h suggested by Fig 5. Such nuclei did indeed appear often, and not exclusively in mature cells where many nuclei could have finished multiplying already (Fig 6A). In relatively young P. falciparum cells with ≤6 nuclear masses, ~10% of them were arrested for at least 5h (Fig 6B). In fact, this proportion fell to ~5% in >6-nucleus schizonts, but the corresponding proportion of entirely unlabelled nuclei rose (i.e. those that had ceased to replicate DNA even before the EdU pulse). In most individual cells, the number of arrested (EdU-only) nuclei was relatively low, 1–2 per cell, but rare cells were observed with more than half of their nuclei arrested for at least 5h (Fig 6C).

This phenomenon was more prominent and occurred more at earlier times in P. knowlesi (Fig 6D–6F). Even in relatively young schizonts, ~15% of nuclear masses were unlabelled–i.e. they had ceased to replicate DNA before the EdU pulse (see Fig 6E: ~15% of nuclei were arrested in cells with 2–6 nuclear masses, and similarly even in cells with 2–3 nuclear masses (S9 Fig)). By contrast, far fewer nuclei were labelled with BrdU only, meaning that they were unlikely to have paused throughout the EdU pulse and then commenced DNA replication again later on–which was a very common observation in P. falciparum. Instead, nuclei in P. knowlesi could either arrest long-term very early in schizogony, or go on to multiply several times with only short pauses between replicative rounds. This supports the observation from Fig 5 that the gaps between replicative rounds were shorter in P. knowlesi than P. falciparum.

Single-molecule DNA replication dynamics do not differ significantly between P. falciparum and P. knowlesi

We previously measured the single-molecule dynamics of DNA replication in P. falciparum, determining that replication fork speed was ~1.2kb/min and the spacing between adjacent replication origins was ~65kb [9]. Since the genome composition of P. knowlesi is very different, we sought to establish whether these parameters would differ in a less A/T-biased Plasmodium genome.

Our previous work employed the gold-standard DNA combing technique [19, 20], which linearises single DNA fibres on glass slides at a uniform stretching factor of 2kb/μm. Cells are first pulse-labelled with two modified nucleosides, CldU and IdU, to generate DNA fibres in which the speed and directionality of replication forks can be measured (Fig 7A) [21]. Unfortunately this technique has recently become unavailable due to the discontinuation of the only antibody that reliably distinguishes CldU and IdU (previously used by all researchers in this field). The alternative double-label, EdU/BrdU, as used here in Figs 2–6, proved incompatible with DNA combing because EdU click chemistry gave poor results on the sialinized glass coverslips specifically required for DNA combing. Therefore, we reverted to the simpler technique of spreading DNA fibres from crude cell lysates on non-sialinized glass slides [22, 23]. This permitted EdU/BrdU labelling (Fig 7B), albeit at the expense of a uniform DNA stretching factor and the loss of very long fibres. DNA spreading tends to generate bundled fibres with only short lengths of single-stranded DNA, particularly when using parasite cells which have haemozoin debris in the cell lysate.

Fig 7C shows that replication fork speeds were not significantly different in the two species, although they trended faster in P. knowlesi. Replication origin spacing was identical in the two species (Fig 7D). Using the published DNA stretching factor of 2.59kb/μm, calculated historically on DNA spreads from human cells [23], replication fork speeds could be calculated at 0.6–0.7kb/min, and inter-origin spacing at ~30kb, but these figures are lower than our earlier measurements on combed DNA fibres from P. falciparum [9], and are probably less accurate because the DNA spreading method is less controlled. DNA in haemozoin-rich Plasmodium lysates may have a fundamentally shorter stretching factor than DNA in human cell lysates, due to, e.g., different viscosity, and measurements from DNA spreads are also skewed to include only the shorter tracts in a population, because longer tracts are more likely to enter a DNA bundle and thus be excluded–a problem that does not arise on long, single, combed DNA fibres. Nevertheless, our measurements are comparable within the conditions used here. The clear result was that DNA replication parameters did not differ significantly in P. falciparum versus P. knowlesi, although there was a trend towards faster replication fork speeds in P. knowlesi. This trend is corroborated by the observation that in a whole-cell context, briefer pulse-labels could be detected in P. knowlesi than in P. falciparum (S6 Fig).

Discussion

This study is the first detailed analysis of DNA replication dynamics throughout schizogony in two different malaria parasites, P. falciparum and P. knowlesi. It reveals some key differences between the two species, such as different periods of active DNA replication and shorter gap phases between successive nuclear multiplications in P. knowlesi than in P. falciparum. There were also similarities: for example, S-phase occupied a similar proportion of the differing cell-cycle lengths in both species.

S-phase occupied ~30% of each cell cycle, i.e. 15h in P. falciparum and 9h in P. knowlesi. During this period, the nuclei in a P. knowlesi schizont completed a maximum of 4 and a mean of 2–3 replicative rounds, whereas P. falciparum completed a maximum of almost 5 and a mean of 3–4 replicative rounds. P. falciparum was therefore proportionally less efficient, averaging an extra 40–45 minutes per nuclear multiplication. This was primarily due to longer gap phases between replicative rounds, since the genomes are very similar in size (~23 and 24Mb), single-molecule dynamics were not strikingly different in the two species, and active DNA replication periods were not consistently faster in P. knowlesi–rather, they were slower in the first replicative round, and then faster in later schizogony.

There are several explanations for the longer pauses between replicative rounds in P. falciparum. This genome may be particularly challenging to replicate, with its high A/T-richness and prevalence of homopolymer tracts leading to errors that require resolution before the chromosomes can be separated. If this is true, a ‘G2 checkpoint’ (well-characterised in mammalian cells but uncharacterised in Plasmodium) must exist. The gap after karyokinesis but before the next replicative round was also longer in P. falciparum and this could be imposed, for example, by a certain amount of polymerases or nucleotides being required before a nucleus can trigger DNA replication, and this threshold being slower to achieve in P. falciparum than in P. knowlesi. Interestingly, a ‘limiting factor’ hypothesis has also been proposed from orthogonal recent work that is discussed in more detail below [11].

During S-phase the number of genomes in a schizont dramatically increases, but surprisingly there was no set-point in the proportion of genomes that could replicate DNA at once. Schizonts were observed with anything from 20% to 100% of their nuclear masses replicating in a 30-minute window, with the average proportion being higher in P. falciparum than in P. knowlesi. Thus, the supply of DNA replication factors must keep pace with the expanding number of genomes–indeed, replicative rounds actually grew faster not slower. The growing anabolic challenge for an unknown ‘limiting factor’ may, however, be one reason why the gaps between replicative rounds increased as schizogony progressed. It may also account for why some nuclei appeared to arrest for much longer than average (either within DNA replication or at anaphase). A second explanation for arrested nuclei would be unresolvable DNA damage, triggering a checkpoint arrest, although there is little evidence as yet for conventional replication checkpoints in this system. (Halogenated nucleotides can also cause light-sensitive DNA damage, but cultures were kept in the dark at all possible times to minimise this risk.)

Both species were strikingly variable in the number of daughter cells produced per schizont. Such variation has been observed repeatedly in P. falciparum, with mean merozoite numbers of 15–22 reported in different strains [12, 17, 24, 25]. This cannot be entirely due to asynchrony in cultured cells: for example, the majority of P. knowlesi cells entered S-phase within a single hour, yet nuclear numbers were extremely variable at 31hpi, when just 2h later the great majority of these schizonts would burst. (Asynchronous S-phase entry may play a somewhat greater role in P. falciparum, where the pre-S-phase growth period was 50% longer and S-phase entry was less tight.) One key explanation for variable schizont size could be heterogenous long-term stalling of some nuclei, as shown in Fig 6: if, at the 2n stage, one nucleus arrests, then the schizont might complete only half as many nuclear multiplications and this would logically lead to highly variable schizonts. Another explanation is inherent variability in host erythrocytes, with young or old cells providing more or less hospitable environments for parasite multiplication. (It was also notable that human erythrocytes from different donors, which can differ in age and many other factors, had a strong impact on cell-cycle length, particularly in P. knowlesi, so cell-cycle length can certainly be influenced by host as well as parasite factors.) Regarding parasite factors, different strains of P. falciparum are known to have characteristically variable cycle times, ranging from ~48h in 3D7 [26] to 42-44h in FCR3 [2]. In this work, only the 3D7 strain was studied.

Our data are largely consistent with data from Ganter and colleagues, who recently characterised S-phase dynamics in P. falciparum in a complementary way, using live-cell microscopy with the DNA replication factor PCNA[11]. Their method is independent of DNA labelling, and therefore of any associated artefacts such as DNA photo-damage, yet the S-phase parameters measured were strikingly similar. They reported that in early schizonts active DNA replication (marked by the presence of PCNA) takes ~40–50 mins per nucleus, with a broad range in different nuclei and a strong trend towards longer periods in the first replicative round. The gaps between rounds, both before and after karyokinesis, were much longer that DNA replication itself, and were longer at the 1-to-2n stage (~75 mins before and ~50 mins after karyokinesis) than at the 2-to-4n stage. Cells beyond this stage were not analysed. These data are broadly consistent with ours: that early schizonts tend to take 40–75 minutes per active replicative round and ~3h in total between successive nuclear multiplications. They do not address our additional finding that gaps become longer in mature schizonts, but these authors did generate a mathematical model for overall schizogony, which demanded a slowing of ~17% in each replicative round after the second. To comply with this, if DNA replication itself successively speeds up then gap phases must become longer: consistent with our data, and also with a requirement to build up a putative limiting factor. Ganter and colleagues also proposed this from their independent dataset, strongly supported by their observation that when several nuclei replicated their DNA synchronously, they took longer to complete.

It was striking that centrin foci, representing the centriolar plaques that act as microtubule organising centres for each genome division, were only detected with ~50% of all nuclear masses in schizonts of either species. These data may have been affected by use of a sub-optimal commercial antibody [18], but nevertheless it is likely that centriolar plaques are not static, but are elaborated and disassembled in at least some components with each replicative round. They clearly disappeared in late schizonts after the final round of karyokinesis, consistent with Simon et al. [18] and expected if the structures get disassembled when no longer required. Furthermore, only a small minority of nuclear masses showed two well-separated plaques, suggesting that their duplication and separation is a late event in the process of DNA replication and karyokinesis. A caveat here is that centriolar plaques were detected only by centrin, which may be one dynamic component of an underlying, static structure. The detailed organisation of the Plasmodium centriolar plaque remains under active investigation [18].

Although Plasmodium schizogony is a very different process from binary fission in mammalian cells, the intra-nuclear organisation of genome replication appears to be similar. Mammalian nuclei show foci of active DNA replication called ‘replication factories’. In early S-phase, when euchromatin is predominantly replicating, factories are small, numerous and dispersed throughout the nucleus; later they appear as larger clumps in the nuclear periphery, representing replicating heterochromatin [27]. Plasmodium nuclei are comparatively tiny but it was nevertheless possible to see brighter foci of newly replicated DNA in the nucleoplasm, resembling replication factories. (These were particularly evident in nuclei stained with BrdU, which was detected less strongly than EdU, using an antibody rather than click-chemistry, thus emphasising the strongest foci.) A small proportion of nuclei showed very clear perinuclear foci, possibly because they were captured while replicating discrete perinuclear clusters of heterochromatin, and some showed DNA replication in only one section of the nucleoplasm–these were much commoner in P. falciparum than P. knowlesi. Chromosomes may be organised differently, or heterochromatin may be more strongly clustered, in P. falciparum, reflecting the well-characterised clustered heterochromatin that encodes var virulence genes [28]. Finally, during the presumptive final round of DNA replication, there was a clear pattern of homogenous but faint staining in all nuclei: this was particularly dominant in P. falciparum. It suggests that the final round is indeed distinct: DNA replication in all nuclei may slow down, either to ensure synchrony in the final karyokinesis, or because a certain factor becomes limiting. Such low-level DNA synthesis could, alternatively, represent post-replication DNA repair, but this seems unlikely because it was usually seen throughout the nucleoplasm of all nuclei, and quite specifically in late schizonts.

At the single-molecule level, there was little difference in the dynamics of polymerase movement (i.e. replication fork velocity) through the genomes of P. falciparum and P. knowlesi, although there was a trend towards faster movement in P. knowlesi. This could be due to genome composition, or to a higher average level of DNA replication factors (proteins or nucleotides) in P. knowlesi schizonts. DNA replication dynamics do frequently differ between different species and even between different human cell lines [29]. Here, however, it seems that the main differences between P. falciparum and P. knowlesi occur at a higher level–i.e. the timing of multiplication for whole nuclei and their associated gap phases–rather than at the level of individual DNA replication forks. It is interesting to speculate the wide variation in replication fork rates may originate partly from pooling cells in which both many and few nuclei are replicating simultaneously, and accordingly where a ‘limiting factor’ is more or less limiting per replication fork.

Overall, this study provides novel detail about the dynamics of schizogony in two different human malaria parasites, and sets the stage for future work to examine how schizogony changes with changing conditions in the human host. A recent publication reported that nutrient limitation in a murine host can markedly reduce the number of progeny per schizont, and suggested that the same may occur in human malaria patients [30]. This may be only one of many host conditions that could influence the growth of malaria parasites, with potentially important impacts on clinical outcome.

Methods

Parasite culture and transfection for ectopic expression of thymidine kinase

P. falciparum parasites were maintained in vitro in human O+ erythrocytes at 4% haematocrit in RPMI 1640 medium supplemented with 25mM HEPES (Sigma-Aldrich), 0.25% sodium bicarbonate, 50 mg/L hypoxanthine standard procedures [31]. P. knowlesi parasites were maintained similarly, maintained at 2% haematocrit instead of 4% and supplemented with 22.2 mM glucose and 10% horse serum instead of human serum.

The P. falciparum 3D7 strain that expresses thymidine kinase has been previously described [10]. A similar thymidine-kinase-expressing P. knowlesi strain was created by transfecting the same plasmid into the A1-H.1 strain, essentially as previously described [32]. Late-stage P. knowlesi parasites were enriched using Histodenz and 10 μl of schizonts mixed in a transfection cuvette (Lonza) with 100 μl of P3 solution (Lonza) containing 30 μg of plasmid. Transfection was carried out using program FP158 (Amaxa Nucleofector, Lonza), followed by immediate transfer into 500 μl of complete culture media mixed with 190 μl uninfected erythrocytes. The transfection mix was incubated at 37°C while shaking at 800 rpm in a thermomixer for 30mins, before being transferred into a 6-well plate, gassed and incubated for one parasite life cycle. Selection was then applied with 100 nM pyrimethamine (Santa Cruz Biotechnology Inc) and daily media changes for 3 days, then routine maintenance until transgenic parasites appeared.

Synchronisation for timecourse experiments

Mature schizont cultures at >6% parasitaemia were synchronised using 55% Nycodenz (Alere technologies AS) [33]. Cultures were centrifuged and media removed to leave 2ml of media and blood, which was layered gently on top of 5 mL of prewarmed 55% Nycodenz, then centrifuged at 1300g for 5 mins. The floating schizont layer was collected and added to a wash buffer containing incomplete RPMI, 4% haematocrit for the final culture volume, and 1.5 μM ‘Compound 2’ (4-[7-[(dimethylamino)methyl]-2-(4-fluorophenyl)imidazo[1,2-a]pyridine-3-yl]pyrimidin-2-amine) [34]), centrifuged (800g for 5 mins), resuspended in complete RPMI and 1.5 μM Compound 2 and incubated at 37°C for 2 h. Cultures were centrifuged (800xg for 5 mins) and supernatant removed. Cultures were washed in prewarmed incomplete media and resuspended in complete RPMI to allow reinvasion. Cultures were split into 5ml aliquots in 50ml falcon tubes and placed in an orbital shaker at 37°C for 1 h to increase reinvasion rate. After reinvasion, the cultures were pooled together, centrifuged and Nycodenz treated again, now retaining the bottom layer containing newly reinvaded ring stages. This layer was washed in incomplete media and resuspended in compete RPMI, marking timepoint 0 hours post invasion (hpi).

Pulse labelling with modified nucleotides

Cultures for single-labelled timecourses were pulse-labelled with 10 μM ethyl-deoxyuridine (EdU) for 30 mins at 1 h intervals. Cultures for double-labelling experiments were first pulse-labelled with 10 μM EdU for 30 mins at specific timepoints, then washed twice in prewarmed incomplete RPMI and resuspended in complete media that was incubated alongside the cultures throughout the experiments, to avoid any disturbance of cell cycle dynamics caused by switching into fresh media. At specific timepoints, cultures were labelled with a second nucleotide, 5-bromo-2’-deoxyuridine (BrdU), at 200 μM for 30 mins at 37°C. Immediately after this label, blood smears were made, air dried, fixed in 2% paraformaldehyde for 5 mins, washed in PBS, washed in dH₂0, air dried and stored at 4°C.

Immunofluorescence

All slides were incubated in 0.2% Triton X-100 for 15 mins and washed in PBS for 5 mins.

Single-nucleotide EdU-labelled slides were incubated in blocking solution (1% BSA in PBS) for 30 mins and then 1:100 anti-centrin antibody (clone 20H5, Millipore) in blocking solution for 1 h at room temperature. Slides were washed three times in blocking solution. EdU signal was detected with click chemistry. Slides were incubated in click reaction buffer (0.845mM Tris HCl pH 8.8, 1mM CuSO₄, 2.5 μM Alexa Fluorescent Azide 594, freshly dissolved 75mM ascorbic acid) for 1 h at room temperature. Slides were washed in blocking solution three times and then incubated with the secondary antibody for centrin detection, goat anti-mouse Alexa 488 (Molecular Probes) for 1 h at room temperature. Slides were washed twice in PBS, incubated with 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 10 mins, washed in PBS, mounted using 20 μl Prolong Diamond Antifade (Molecular Probes) and set overnight at room temperature.

Double-labelled EdU/BrdU slides were incubated with 0.2% Triton-X for 15 mins, washed in PBS for 5 mins, incubated in 1M HCl for 1 h and washed in PBS for 5 mins. EdU labels were “clicked” as above. Slides were washed with PBS three times. Slides were then incubated with 20 mM non-fluorescent dye Azidomethylphenosulphide (Sigma) for 30 mins at room temperature to block remaining EdU residues. Slides were incubated with blocking solution for 30 mins. Primary immuno-detection of BrdU was with rat anti BrdU BU1/75 (ICR1) antibody (1:100 dilution, Abcam). Slides were washed in blocking solution three times and incubated with goat anti-rat Alexa 488 secondary antibody (1:500 dilution, Molecular probes) at room temperature for 1 h; washed, incubated with DAPI and mounted as above.

Data analysis and statistics

Slides were imaged using a Nikon Microphot SA microscope equipped with a Qimaging Retiga R6 camera and a 100X oil objective (Leica, 1.30 na). 100 parasites were counted for each timepoint of the single-nucleotide-labelled timecourses, and 50 parasites were counted for double-labelled timecourses. For the ‘stalling’ experiment, 20 parasites were counted for each category of schizont maturity. Images were classified regarding presence and number of centrin foci and the presence and pattern of nucleotide labelling (BrdU/EdU) within the nucleus. Wide-field microscopy was used throughout because very large numbers of parasite images were required, accepting that the size of individual nuclear masses approaches the limit of resolution for simple light microscopy. (See S2B Fig for a comparison of single-projection versus confocal microscopy when used for counting nuclear masses. Confocal imaging was carried out using a Zeiss LSM 700 microscope using Zen10 software). Data were plotted using Graphpad Prism and the statistical significance of differences between groups of data was calculated via Mann Whitney tests or analysis of variance.

DNA fibre spreading

DNA fibre spreading was performed as previously described [22]. Cultures were labelled with 10μM EdU for 10 mins, then 100 μM BrdU for 10 mins. 2μl of saponin-released parasites was pipetted near the top of a glass slide and allowed to dry for ~5 mins, until sticky but not dry. 7 μl of spreading buffer (20 mM TrisHCl pH 7.4, 50 mM EDTA pH 8, 0.5% SDS) was added and stirred gently with a pipette tip to release the DNA. The slide was incubated for 2 mins and then tilted (15°) to let drop run slowly down the slide, producing a constant stretching factor of 2.59kb/μM [23]. Slides were air dried, fixed in MeOH: acetic acid (3:1), air dried and stored at 4°C.

Detection of EdU and BrdU in DNA fibre spreads

Fibre spreads were denatured with 1M HCl for 75 mins, washed three times in PBS and blocked with blocking solution (1% BSA and 0.1% Tween 20 in PBS). Immuno-detection of double labelled fibre spreads was done with EdU click chemistry and then antibodies diluted in blocking solution, each incubated with a coverslip on top in a humid chamber at room temperature for 1h. Slides were incubated with click reaction buffer as above, followed with three washes in blocking buffer. Slides were then incubated in blocking solution for 30 mins. Primary immuno-detection for BrdU was done with rat anti BrdU BU1/75 (ICR1) antibody (1:100 dilution, Abcam), together with mouse anti ssDNA (clone16-19) antibody (Millipore, 1:300 dilution). The secondary antibodies (Molecular Probes) were goat anti-rat coupled to Alexa 488 (1:500 dilution) and goat anti-mouse coupled to Alexa 405 (1:500 dilution). Slides were washed three times in PBS and mounted using 20μl Prolong Diamond Antifade (Molecular Probes), set overnight at room temperature. Single-labelled fibre spreads were detected above but without click chemistry.

Image Acquisition and Processing of DNA fibre data

Image acquisition was via a Nikon Microphot SA microscope equipped with a Qimaging Retiga R6 camera. Images were acquired with a 100X oil objective (Leica, 1.30 na) where 1 μm = 28.65 pixels, which corresponds to 69.8 bp per pixel (DNA stretching factor 2kb/ μm for DNA combing) and 90.40bp per pixel (DNA stretching factor 2.59kb/μm for DNA spreading). Observation of long DNA fibres required the capture and assembly of adjacent fields. Replication tracts and fibre lengths were measured manually using ImageJ software. Statistical analysis and graphs of BrdU tract length and replication velocities were performed using GraphPad Prism.

Availability of raw data

All data are available in the Dryad repository at doi:10.5061/dryad.ghx3ffbr8 [35].

Supporting information

S1 Fig. DNA replication rates are similar in the newly-generated P. knowlesi TK-expressing line and in the parent line.

Synchronised parasites of both lines were exposed to either or both modified nucleosides at the levels used in subsequent experiments (10 μM EdU, 200 μM BrdU) for a 6h period covering the majority of S-phase. DNA content was then measured via SYBR-green 1 DNA dye, as previously published [36], in triplicate, and expressed as fold-change in DNA content from the start of the experiment. No significant difference in growth rates was observed in either line, exposed or not exposed to modified nucleosides.

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S2 Fig

Examples of A) a typical slide, showing 3 parasites classified for their number of nuclear masses, number of centrin foci and presence/absence of EdU staining. B) confocal microscopy versus the wide-field method used throughout this work, showing that confocal yields very similar detection of the number of features per cell.

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S3 Fig. Biological replicates of timecourses shown in Fig 2.

In the early parts of the timecourses, samples were taken every 3h for P. falciparum and every 2h for P. knowlesi, rather than every hour, as in Fig 2. In the final parts of both timecourses, samples were again taken every hour. Final timepoints in both timecourses show reinvasion, i.e. high proportions of 1n cells. A, B: Scatter graphs of nuclear numbers in P. falciparum (A) or P. knowlesi (B), n = 30 cells, medians are shown in red. C, D: Percentage of 30 cells showing some EdU labelling at each timepoint in P. falciparum (C) and P. knowlesi (D).

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S4 Fig. Data from Fig 3D (i.e. intensity of EdU staining seen at each timepoint in P. falciparum and P. knowlesi) shown as individual graphs for each category of staining defined in Fig 3B.

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S5 Fig

A: Schematic showing the process of karyokinesis that has previously been proposed in Plasmodium, highlighting the role of the centriolar plaque (basis outlined in Gerald et al. [13]). B: Examples of the distinct patterns of centrin staining seen on the highlighted nuclear masses: no foci, a single focus, 2 adjacent foci, 2 opposite foci. P. falciparum are shown, representative of pattern in both species. Scale bar all panels 1μm. C: Percentage of nuclear masses with 0, 1 or 2 centrin foci throughout schizogony, n = 100. D: Percentage of nuclear masses with 0, 1 or 2 centrin foci that also showed or did not show active DNA replication (EdU staining) within the previous 30mins. E: Percentage of nuclear masses with 0, 1 or 2 centrin foci and patterns of intranuclear DNA replication (full, partial, or discrete foci) throughout schizogony. F: Data as in Data as in A, replotted by number of by number of nuclear masses per cell rather than hpi.

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S6 Fig. The minimum pulse-labelling period that can be detected via EdU-labelled DNA was tested.

A: In P. falciparum, 3 minutes was detectable (faintly) and 5 minutes gave reasonably bright signal. In P. knowlesi, labelling was clearly detectable within 1 minute. B: Examples of the appearance of stained nuclei when cells are simultaneously labelled with EdU and BrdU. Scale bar all panels 2μm.

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S7 Fig. Biological replicate of the experiment shown in Fig 4, measuring the length of the first replicative round in P. falciparum and P. knowlesi cells.

Graphs show the percentage of nuclei in 1n cells that labelled with EdU and BrdU (n = 20). Scatter plots display the same data broken down per-cell, with means shown.

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S8 Fig

A: Schematic of the timecourses shown in this figure. B: Representative examples of cells across the double-labelled timecourse with 2h intervals for P. falciparum. Scale bar all panels 2μm. B: Percentages of nuclear masses labelled with EdU alone, BrdU alone, or both labels throughout the timecourses shown in (B).

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S9 Fig. Data as in main Fig 6, showing the percentages of P. knowlesi nuclei labelled with EdU and BrdU, EdU alone, BrdU alone, or neither label (n = 20 cells), after both 2h and 5h –demonstrating a higher percentage of arrested nuclei after 2h (~22% of S-phase) than 5h (>50% of S-phase), but an overall similar picture.

Data are also stratified into cells with 2–3, 4–6, or more than 6 nuclear masses, showing that arrested nuclei are still detected in very young 2-3n schizonts.

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Acknowledgments

We are grateful to the lab of Prof. Julian Rayner for making the initial thymidine kinase transfectant in P. knowlesi, to Dr Holly Craven for help with revisions, and to Dr Francis Totanes and Dr Andrew Blagborough for critical reading of the manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by a European Research Council (https://erc.europa.eu/) Research grant, ‘Plasmocycle’ (725126) to CJM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Striepen B, Jordan CN, Reiff S, van Dooren GG. Building the perfect parasite: cell division in apicomplexa. PLoS pathogens. 2007;3(6):e78. doi: 10.1371/journal.ppat.0030078 ; PubMed Central PMCID: PMC1904476. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Arnot DE, Ronander E, Bengtsson DC. The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony. International journal for parasitology. 2011;41(1):71–80. doi: 10.1016/j.ijpara.2010.07.012 . [DOI] [PubMed] [Google Scholar]
3.Wassmer SC, Taylor TE, Rathod PK, Mishra SK, Mohanty S, Arevalo-Herrera M, et al. Investigating the Pathogenesis of Severe Malaria: A Multidisciplinary and Cross-Geographical Approach. Am J Trop Med Hyg. 2015. doi: 10.4269/ajtmh.14-0841 . [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, et al. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med. 2005;2(8):e204. doi: 10.1371/journal.pmed.0020204 ; PubMed Central PMCID: PMC1188247. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Branco A, Melo-Cristino J. Extreme parasitemia in P falciparum malaria. Blood. 2018;132(8):868. doi: 10.1182/blood-2018-07-861880 . [DOI] [PubMed] [Google Scholar]
6.Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419(6906):498–511. doi: 10.1038/nature01097 . [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, et al. Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature. 2008;455(7214):757–63. doi: 10.1038/nature07327 ; PubMed Central PMCID: PMC2651158. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Pain A, Bohme U, Berry AE, Mungall K, Finn RD, Jackson AP, et al. The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature. 2008;455(7214):799–803. doi: 10.1038/nature07306 ; PubMed Central PMCID: PMC2656934. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Stanojcic S, Kuk N, Ullah I, Sterkers Y, Merrick CJ. Single-molecule analysis reveals that DNA replication dynamics vary across the course of schizogony in the malaria parasite Plasmodium falciparum. Sci Rep. 2017;7(1):4003. doi: 10.1038/s41598-017-04407-z ; PubMed Central PMCID: PMC5479783. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Merrick CJ. Transfection with thymidine kinase permits bromodeoxyuridine labelling of DNA replication in the human malaria parasite Plasmodium falciparum. Malar J. 2015;14(490). doi: 10.1186/s12936-015-1014-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Klaus S, Binder P, Kim J, Machado M, Funaya C, Schaaf V, et al. Asynchronous nuclear cycles in multinucleated Plasmodium falciparum facilitate rapid proliferation. Sci Adv. 2022;8(13):eabj5362. doi: 10.1126/sciadv.abj5362 . [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Reilly HB, Wang H, Steuter JA, Marx AM, Ferdig MT. Quantitative dissection of clone-specific growth rates in cultured malaria parasites. International journal for parasitology. 2007;37(14):1599–607. doi: 10.1016/j.ijpara.2007.05.003 ; PubMed Central PMCID: PMC2268714. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Gerald N, Mahajan B, Kumar S. Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryotic cell. 2011;10(4):474–82. doi: 10.1128/EC.00314-10 ; PubMed Central PMCID: PMC3127633. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Francia ME, Striepen B. Cell division in apicomplexan parasites. Nat Rev Microbiol. 2014;12(2):125–36. doi: 10.1038/nrmicro3184 . [DOI] [PubMed] [Google Scholar]
15.Rudlaff RM, Kraemer S, Marshman J, Dvorin JD. Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis. PLoS pathogens. 2020;16(6):e1008587. doi: 10.1371/journal.ppat.1008587 ; PubMed Central PMCID: PMC7302870. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Malinsky J, Koberna K, Stanek D, Masata M, Votruba I, Raska I. The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase. Journal of cell science. 2001;114(Pt 4):747–50. doi: 10.1242/jcs.114.4.747 . [DOI] [PubMed] [Google Scholar]
17.Simon CS, Sturmer VS, Guizetti J. How Many Is Enough?—Challenges of Multinucleated Cell Division in Malaria Parasites. Front Cell Infect Microbiol. 2021;11:658616. doi: 10.3389/fcimb.2021.658616 ; PubMed Central PMCID: PMC8137892. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Simon CS, Funaya C, Bauer J, Vobeta Y, Machado M, Penning A, et al. An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites. Life Sci Alliance. 2021;4(11). doi: 10.26508/lsa.202101199 ; PubMed Central PMCID: PMC8473725. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Michalet X, Ekong R, Fougerousse F, Rousseaux S, Schurra C, Hornigold N, et al. Dynamic molecular combing: stretching the whole human genome for high-resolution studies. Science (New York, NY. 1997;277(5331):1518–23. doi: 10.1126/science.277.5331.1518 . [DOI] [PubMed] [Google Scholar]
20.Lebofsky R, Bensimon A. Single DNA molecule analysis: applications of molecular combing. Brief Funct Genomic Proteomic. 2003;1(4):385–96. doi: 10.1093/bfgp/1.4.385 . [DOI] [PubMed] [Google Scholar]
21.Schwob E, de Renty C, Coulon V, Gostan T, Boyer C, Camet-Gabut L, et al. Use of DNA combing for studying DNA replication in vivo in yeast and mammalian cells. Methods Mol Biol. 2009;521:673–87. doi: 10.1007/978-1-60327-815-7_36 . [DOI] [PubMed] [Google Scholar]
22.Parra I, Windle B. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature genetics. 1993;5(1):17–21. doi: 10.1038/ng0993-17 . [DOI] [PubMed] [Google Scholar]
23.Jackson DA, Pombo A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. The Journal of cell biology. 1998;140(6):1285–95. doi: 10.1083/jcb.140.6.1285 ; PubMed Central PMCID: PMC2132671. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Garg S, Agarwal S, Dabral S, Kumar N, Sehrawat S, Singh S. Visualization and quantification of Plasmodium falciparum intraerythrocytic merozoites. Syst Synth Biol. 2015;9(Suppl 1):23–6. doi: 10.1007/s11693-015-9167-9 ; PubMed Central PMCID: PMC4688406. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Stewart LB, Diaz-Ingelmo O, Claessens A, Abugri J, Pearson RD, Goncalves S, et al. Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites. Commun Biol. 2020;3(1):624. doi: 10.1038/s42003-020-01349-7 ; PubMed Central PMCID: PMC7595149. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Bell DR S.; Patel A.P.; Lee S.H.; Chang Y.T.; Deu E. High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle. BioRxiv. 2021;doi.org/10.1101/2021.03.15.434938 Epub 2021. [Google Scholar]
27.Dimitrova DS, Gilbert DM. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat Cell Biol. 2000;2(10):686–94. doi: 10.1038/35036309 ; PubMed Central PMCID: PMC1255923. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Freitas-Junior LH, Bottius E, Pirrit LA, Deitsch KW, Scheidig C, Guinet F, et al. Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature. 2000;407(6807):1018–22. doi: 10.1038/35039531 . [DOI] [PubMed] [Google Scholar]
29.Techer H, Koundrioukoff S, Azar D, Wilhelm T, Carignon S, Brison O, et al. Replication dynamics: biases and robustness of DNA fiber analysis. Journal of molecular biology. 2013;425(23):4845–55. doi: 10.1016/j.jmb.2013.03.040 . [DOI] [PubMed] [Google Scholar]
30.Mancio-Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, et al. Nutrient sensing modulates malaria parasite virulence. Nature. 2017;547(7662):213–6. doi: 10.1038/nature23009 ; PubMed Central PMCID: PMC5511512. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Trager W, Jensen JB. Human malaria parasites in continuous culture. Science (New York, NY. 1976;193(4254):673–5. doi: 10.1126/science.781840 . [DOI] [PubMed] [Google Scholar]
32.Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(2):531–6. doi: 10.1073/pnas.1216457110 ; PubMed Central PMCID: PMC3545754. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Mohring F, Hart MN, Patel A, Baker DA, Moon RW. CRISPR-Cas9 Genome Editing of Plasmodium knowlesi. Bio Protoc. 2020;10(4):e3522. doi: 10.21769/BioProtoc.3522 ; PubMed Central PMCID: PMC7842605. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Donald RG, Zhong T, Wiersma H, Nare B, Yao D, Lee A, et al. Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Molecular and biochemical parasitology. 2006;149(1):86–98. doi: 10.1016/j.molbiopara.2006.05.003 . [DOI] [PubMed] [Google Scholar]
35.McDonald J, Merrick CJ. DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi. 2022. Dryad Digital Repository. doi: 10.5061/dryad.ghx3ffbr8. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrobial agents and chemotherapy. 2004;48(5):1803–6. doi: 10.1128/AAC.48.5.1803-1806.2004 ; PubMed Central PMCID: PMC400546. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Pathog. doi: 10.1371/journal.ppat.1010595.r001

Decision Letter 0

Xin-zhuan Su, Michael J Blackman

14 Feb 2022

Dear Catherine,

Thank you very much for submitting your manuscript "DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email) and following extensive consideration, we would like to give you the opportunity to submit a significantly-revised version that addresses or takes into account the reviewers' comments.

As you will see, the reviewers reached widely divergent views on the manuscript. Reviewers 1 and 2, however, raised significant concerns that need to be addressed before the work can be considered further. In particular, questions were raised over the impact of BrdU and EdU labelling on cell cycle progression in the parasite lines, the significance of the apparent loss of centrin signal from nuclei, the number of experimental biological replicates used to draw several of the key conclusions, and whether the light microscopic system used provides sufficient resolution to reach some of the conclusions drawn. We share these concerns. All the reviewers additionally raised a number of additional points that should be addressed.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael J Blackman

Associate Editor

PLOS Pathogens

Xin-zhuan Su

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors provide an interesting description of replication dynamics throughout erythrocytic schizogony of two Plasmodium species. The amount of data generated is remarkable and the used technology is interesting. However, the study remains descriptive, and the draw conclusion do not extend significantly enough upon the current understanding of replication. The gain of sustainable insight and the ability to generate new hypotheses is a bit too limited to warrant a publication in a journal like PLoS Pathogens. Further, there are some important technical concerns that would have needed to be more thoroughly addressed by the authors to consolidate their data. Lastly the manuscript itself suffers from a difficulty to understand the individual experimental parameters and outcomes. Given some improvements I, however, know that this study can find its place in another journal. In the following I want to outline my criticism in more detail to help the authors to improve the manuscript.

Reviewer #2: In a patient infected with Malaria, Plasmodium parasites replicate in red blood by an atypical cell division mode called schizogony. Multiple rounds of asynchronous DNA replication and nuclear fission generate a multinucleated cell and, specialized cytokinesis named segmentation produces 10 to 30 daughter parasites depending on the Plasmodium species. As McDonald et al. highlighted in their study, the rate and efficiency of parasites replication are fundamentally critical to malaria pathogenesis. In the case of malaria infection by P. falciparum, peripheral parasitemia will determine the severity of the disease. To better understand the molecular mechanisms regulating the number of daughter parasites, the study's authors took a very original and ambitious approach by studying schizogony in two different human Plasmodium parasites, falciparum, and knowlesi, that differ in their cell cycle length and the number of progeny. The authors successfully generated parasite lines that allow de novo DNA replication to be followed by incorporating pulse labels of modified nucleotides. The authors performed different time courses using single EdU pulse labels or sequential pulses of two nucleotides (EdU and BrdU). All experiments readouts were done by light microscopy, except for the determination of the replication fork speed on spreading DNA fibers from cell lysates on glass slides. Unfortunately, I have significant concerns regarding the experimental robustness of the study, where it appears that for all data presented in the study, there is only a single biological replicate. In addition, the authors' microscopy technique does not provide the required resolution to resolve two nuclei, especially in the z-axis when they analyze multinucleated with more than four nuclei. Therefore the authors can observe and measure DNA replication per cell but not per nucleus.

Furthermore, they wrongly interpreted the published data on the MTOC dynamics in Simon et al.'s study, leading them to inappropriately conclude that 50 % of nuclei lack centrin staining, likely representing false negatives. In conclusion, I have raised significant comments that need to be addressed, and numerous statements must be revised based on the limitation of the experimental settings. In addition, I made some minor comments to help the authors identify sections of the study that remain unclear to me.

Reviewer #3: This work offers a detailed and comparative analysis of DNA replication dynamics of two distinct malaria parasite species. P. knowlesi and P. falciparum. It is of particular importance as the first paper for which the replication dynamics of Pk are described, and uses a series of neat approaches to compare the specific replication parameters. This comparison is particularly important as the two parasites have significant differences in cycle length (Pf 2x as long) and AT-content, as well as ultimately producing differing numbers of progeny merozoites. The studies revealed surprising similarities in the single molecule speed of replication as well as inter origin distance, but some clear differences in whole cell replication dynamics – with a surprisingly synchronous onset of S phase and shorter gap phases.

Although descriptive the data presented are of fundamental importance to understanding these processes, and thus I would say they are both highly important for work in the field, and would provide significant interest to the pathogen research community. The paper is very well written and the conclusions are well supported by the data. In the current version, it is slightly unclear as to what biological repeats have been carried out for each experiment, and this is something that will need to be clarified. The other issues raised are relatively minor, although efforts to examine perturbations of these dynamics would add another level of interest.

Rob Moon

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The authors don´t comment sufficiently on the very low mean number of nuclei at the end of schizogony (P.f. 11 and P.k. 6) shown in Fig. 2. This stands in contrast to what has been measured in several studies by e.g. Garg et al. , Reilly et al., Simon et al. and would warrant a more detailed analysis. The observation that number of nuclei is highly variable is not novel as suggested in the abstract.

The authors claim that “The length of S-phase is proportional to the overall length of erythrocytic schizogony”. I think their data shown in Fig. 2 is informative overall and underpins that schizogony takes about 30% of the cycle. This, however, shouldn’t be mistaken for a correlation between total cell cycle length and schizogony at the single cell level and clarified accordingly.

The authors claim that centrin signal temporarily disappears from a majority of nuclei and suggest this represents a centriolar plaque disassembly. To my knowledge temporary centrosome disassembly (down to centrin) during a division phase is not described in any eukaryote. For Plasmodium specifically multiple studies investigating centrin don’t report any similar findings although their imaging data quality is good (Arnot et al. 2011, Roques et al. 2019, …). Particularly the Simon et al. study referenced by the authors, as far as I understand does only refer to 50% of mononucleated nuclei lacking a centrin signal while in multinucleated stages all nuclei carry one or two centrin foci. Finally, the signal-to-noise ratio shown in Fig. 4B casts significant doubt on the validity of this rather bold claim.

Most importantly, the authors fail to demonstrate that cell cycle progression was “not overtly affected” (line 117) by their labeling. This is particularly critical in the light of what the last author has shown in Merrick et al. 2015 where TK expressing parasites displayed a high sensitivity to BrdU (and possibly EdU?) and a severe growth phenotype at significantly lower concentrations than used in this study (IC50 of 219nM measured for a 1 hour pulse – against - 200µM pulse for 30min used here). Unfortunately, this puts into question the interpretation of a big proportion of the data and the authors have done very little to alleviate this concern. BrdU toxicity could for example be the cause for the replication gaps observed for a subpopulation of nuclei in Fig. 6. This is probably even more critical for the observations made in Fig. 7 that nuclei seem to pause replication for extended periods of time.

Reviewer #2: General

My main concern is that it appears that for all data presented in the study, there is only a single biological replicate, in particular for assays run with P. falciparum. Whether there are multiple biological replicates or only one should be specified for every experiment in the figure legend. Suppose there is only a single biological replicate. In that case, the authors should provide extensive justification for why this is the case, and this should be reflected by significantly dampening the corresponding conclusions. The authors note the difference in observed lifecycle length for P. knowlesi based on different batches of donor blood and in different P. falciparum 3D7 parasite line ranging from ~39 hours to the ~48 hours reported in this study (Wockner et al., 2020 (PMID: 31679015); Duffy and Avery, 2017 (PMID: 28738214). Considering the potential influence of lifecycle length on the estimation of the length of S-phase.

In line 184, the authors rigorously and rightfully used the term DAPI-stained area instead of nucleus. The resolution is calculated by the following formula Resolution=1.22 (λ/(NAobj + NAcond)), if we assume that the authors used an objective with a high numeric aperture or NA (1.45) and a condenser of 0.55, the resolution for the DAPI channel (405 nm) is as best 247.5 nm for the lateral resolution (resolution in the focal plane). Therefore in this study, the resolution is four times bigger than an individual nucleus (200nm at best versus 50 nm); consequently, the authors cannot identify single nuclei in a multinucleated cell and must revise their results statements.

The data from the referenced Simon et al. study are inappropriately referenced to suggest that detection of centrin in ~50% of nuclei is consistent with previous literature. This was only the case in mononucleated parasites with a hemispindle, with the following statement made in that article: “Consistent with the late appearance of PfCentrin1-GFP in our live-cell imaging data, only 24 out of 52 analyzed hemispindles in mononucleated cells were associated with an endogenous centrin signal, whereas in later stages, after the first division, every nucleus was accompanied by one or two centrin foci”. Presumably, from 33-47hpi in P. falciparum and 23-33hpi in P. knowlesi, you have relatively few mononucleated parasites. Therefore the ~50% of nuclei lacking centrin staining likely represents a vast number of false negatives. Finally, the anti-centrin antibody labels only one protein present at the cytosolic face of the MTOC, which is a large multiprotein structure embedded in the nuclear envelope for which we know very few components. The authors should address this and consider this when making conclusions about centrin and MTOC.

Results

Line 115: “We confirmed that this permitted the labelling of DNA replication with modified nucleotides (bromo/chloro/iodo/ethyl-deoxyuridine (BrdU/CldU/IdU/EdU)), and that parasite behaviour in terms of fitness and cell-cycle timing was not overtly disturbed". Data is not shown or unclear where the authors presented it in the manuscript.

Line 133-135: the number of nuclei detected at the latest timepoints for schizonts are drastically lower than previously published numbers of merozoites per schizont, at least for P. falciparum where this has been well characterized (Garg et al., 2015 (PMID: 26702305); (Rudlaff et al., (PMID: 32511279)). The authors should elaborate on why this is the case, be it an issue of resolution, parasite maturity, or something else, and clearly state this when making conclusions based on the number of nuclei per cell.

Line 139: “…the onset of S-phase for the majority of cells was quite synchronous (Figure 2C, D) particularly in P. knowlesi, where ~60% of cells commenced replication within a single hour at 22-23hpi.” This doesn’t seem to be supported by the data in Figure 2C & D. According to the methods, these cultures are synchronized to within 1 hour of each other, yet there is a 4 hour difference between when the first parasites begin to replicate (22hpi) and when the greatest number of parasites are replicating (26hpi). This could be interpreted as up to 4 hours of replication asynchrony within a population that should be within 1 hour of age.

Line 145: “Numbers of nuclei stopped increasing at 46-47hpi, making 15 hours the total S-phase period across the population.” It seems in Figure 2C that ~80% of cells are EdU positive at 47 hours, indicating that at 47 hours, 80% of cells are still replicating and could very well increase in DNA content resulting in the formation of new nuclei past the 47 h.p.i. Considering that 80% of parasites were still replicating at this time point, it is difficult to see how it can be used to conclude the length of the total S-phase. The measure of the total content of DNA would be a more appropriate measure than many nuclei, which again cannot be resolved with the microscopy technique presented in the study without treating the parasite with E64, which would allow to increase in the physical distance between nuclei and allow their counting by light microscopy. Therefore the data presented in Figure 2, panels A, B, C, F, G, and H must be revised and interpreted accordingly.

Line 153-154: “P. falciparum showed substantial reinvasion by 48hpi (Supp Figure 2), and so has a similar window of ~2h between the end of S-phase and reinvasion.” Supp Figure 2 doesn’t appear to have any data regarding EdU labelling, and so can’t be used to support the claim that S-phase has ended.

Line 186-189 “…particularly notable in P. falciparum, was a subset of nuclei with homogenous but fainter staining, suggesting that DNA had replicated much less rapidly than in the common pattern: this appeared mostly in late-stage cells completing their presumptive final round of replication (Figure 3B, see P. falciparum at 44-45hpi, P. knowlesi at 30hpi, and quantification in Figure 3C, D)” There are many reasons why fluorescence intensity may differ between different cells, and it is unclear why DNA replicating more rapidly would be the reason for this difference. Also, your study and others suggest that DNA replication occurs faster at the later stages of schizogony, which contrasts with the idea that DNA had replicated less rapidly in these cells.

Line 210-212: “In both species there was some evidence that nuclei with centrin foci anti-correlated with actively replicating, centrin-negative nuclei see for example 42hpi in Figure 4D). These observations point to a temporal separation between the start of DNA replication and the start of centriolar plaque elaboration and duplication”. Considering the previously mentioned likely high false negative rate, and a lack of statistical tests showing this correlation, the data presented in Figure 4 is not sufficiently robust to support these claims. Additionally, it is unclear how you can infer temporal separation between DNA replication and MTOC without live-cell microscopy.

Line 249: “Therefore, replicative rounds became progressively faster as ploidy increased” In my understanding figure 5 supports the conclusion that DNA replication is asynchronous in both Plasmodium species and that is all. And because the experimental design does not allow live imaging therefore authors cannot assess the rate of round of DNA replication like it has been done in the preprint of Klaus et al prublished in 2021.

Discussion

Line 367: " The growing anabolic challenge may, however, be one reason why the gaps between replicative rounds increased as schizogony progressed. It may also account for why some nuclei appeared to arrest for much longer than average". The authors assess DNA replication in this study and not DNA segregation and karyokinesis. The authors call the arrested nucleus what might be an anaphasic nucleus when the two replicated interpolar microtubules connect DNA masses within the same nuclear envelope upon nuclear fission. It is unknown how long this step takes during plasmodium mitosis and whether or non this process is shorter in time as schizogony progresses.

Line 405: "centriolar plaques are disassembled with each replicative round". This relates to the previous comment regarding centrin not being an appropriate marker for the entire MTOC and the high false-negative rate. Supplementary Video 2 of the Simon et al. study shows first the first ~2-3 rounds of mitosis, where intranuclear microtubules are always associated with at least one centrin focus. Additionally, in another study where MTOC of P. falciparum was observed (using NHS ester instead of centrin), an MTOC was observed for every nucleus for every image included in the study (Liffner & Absalon, 2021 (PMID: 34835432), see supplementary videos).

Figures

While the median is often reported, there are no error bars presented on any of the data in the study. Therefore, the authors should include and define error bars for all data where multiple data points are reported.

Many of the microscopy images presented in this study lack scale bars or only one image in an entire panel of figures will have a scale bar. Therefore, either all images should include defined scale bars. If the imaged area is identical for all images in a particular panel, this should be stated in the figure legend that the same scale bar can be applied to all images.

For Figures 3a and 4b, it is currently unclear which organism these images represent. Therefore, the representative images should include examples of EdU and centrin staining classifications for both P. falciparum and P. knowlesi.

Methods

Based on the synchronization protocol listed in the methods, the authors seem to purify viable ring-stage parasites using Nycodenz. However, the authors do not reference a previous study establishing this protocol. If this protocol was developed in this study, the authors should provide validation data that it purifies ring-stage parasites or provide a reference if developed elsewhere.

The numerical aperture for the objective lens used in this study should be stated.

Reviewer #3: Major Points

The experimental design is laid out in Fig 1, and numbers of cells examined are mentioned in several figures but it is not clear what biological repeats have been carried out. Are cells examined all from the same cultures on same day? Or imaged from different repeat experiments. I would expect some parameters measured to vary depending on how “happy” a given culture was, and so data from independent cultures is vital. Could the authors clarify how biological repeats were undertaken, clearly stating this in each figure.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Generally, I notice a “disconnect” between the findings claimed in the abstract, the sub-titles of the results and figures, and the conclusion made at the end of each sub-chapter of the results. This leads to difficulties in clearly grasping the main findings of the study.

Associating the schematics of the experimental setup directly with the corresponding figure would be very helpful to the reader to easier understand the complex data.

The category “fully stained, less bright” used in Fig. 3 bears some risk of not being clearly separatable from the background. Possibly a more objective criterion could be applied here. Maybe it is also helpful to stratify the integrated densities shown in Fig. 3D by the categories shown in Fig. 3C.

For Fig. 5 it would be good to show a simultaneous labeling with BrdU and EdU as a positive control.

The interpretation of the data collected in Fig. 6 could benefit from a more detailed explanation or visualization. Further, the authors could improve on their argumentation how their pulse labeling protocols distinguishes between replication length, replication gaps, and continuously, or not at all replicating nuclei in a multinucleated population.

The authors use the term “syncytial mode of replication” in the abstract. The word syncytium is, however, clearly defined as a multinucleated cell resulting from cell-cell fusion not from multiple nuclear divisions without cytokinesis (Daubenmire, 1936, Science and Wikipedia). Although I have noticed this term see casually applied for Plasmodium spp. I want to strongly advice against this usage.

Reviewer #2: Introduction

Line 47: “The malaria parasite Plasmodium”. This should be revised to say the genus of malaria parasites.

Line 95: the following is posed as an unanswered question “Do all successive rounds of nuclear replication take the same time?” This question has largely been addressed by the Simon et al., and Klaus et al., studies cited elsewhere in this paper.

I personally appreciate when authors conclude the introduction section by summarizing the major findings of the study, which is missing here.

The following statements should be qualified with references:

Line: 55 “Parasitaemia (Parasitemia) in human malaria can vary greatly, partly because the human-infective Plasmodium species (P. falciparum, P. vivax, P. malariae, P. knowlesi, and P. ovale wallikeri & curtisi) differ in their preferences for human erythrocytes.”

Line 58: “…P. vivax is restricted to invading scares reticulocytes, P. malariae primarily invades older erythrocytes, and P. falciparum invades cells of all…”.

Line 61: “There are also other inter-species differences in the process of schizogony. Its duration varies from ~72 hours in P. malariae to ~28 hours in P. knowlesi and the number of merozoites produced per schizont varies from as many as ~30 in P. falciparum to ~15 in P. knowlesi.”

Line 68: “…P. vivax and P. knowlesi have A/T contents of only 60 and 61%.”

Line 71: “These differ in parameters such as genome content, cell cycle period and merozoite number.”

Line 81: “…the first round of DNA replication begins more than halfway through the cell cycle (which is theoretically 48h, actually ~42-50h in different stains and culture systems).”

Line 85: “Duplication of the centriolar plaque (the Plasmodium centriole-equivalent) has been proposed to initiate each replication.”

Line 98: “At the single-molecule level, our recent work defined the average speed of replication forks and spacing of replication origins in P. falciparum schizogony at 1.2 kb/min and 65kb between origins – broadly similar to parameters in other eukaryotic cells. These averages changed by ~30% over the course of schizogony, with the fastest fork movement and most widely-spaced origins occurring early on.”

Line 102: “The opposite pattern occurs in human cells, where replication speed is usually limited by cellular nucleotide pools and becomes fastest towards the end of S-phase as nucleotide production peaks.”

Results

In the results, it is stated that “…nuclei with the ‘partial’ replication patterns made up 20-30% of the total in P. falciparum at all timepoints, but fewer than 10% of the total in P. knowlesi (Figure 3C)” At the final time point, however, it seems as if the partial replication pattern takes up approximately 30%.

Considering the resolution of the microscopy used in this study and that images were not acquired in 3 dimensions, the authors should consider simplifying the two centrin foci classifications in Figure 4. In 3 dimensions, two centrin foci could be ‘opposite’ to each other but have moved up or down relative to how the viewer sees the image rendered in 2D. Therefore these would appear next to each other but, in reality, be opposite.

The Simon et al. study referenced in the paper quantifies the time P. falciparum nuclei spend with a single or double centrin focus, beginning at about 150 minutes for the first mitosis and continuing at around 100 minutes for subsequent rounds. This observation should be mentioned when discussing the length of time a nucleus has two centrin foci.

The results state that “… nuclei with centrin foci were never seen past the stage of 8-9 nuclei in P. knowlesi (Supp Figure 3).” Despite this, in Supp Figure 3, there are one centrin data points for cells with 10, 11,12, and 15 nuclei.

Treatment of cells with BrdU and EdU induces some DNA damage. For nuclei that have been arrested well before other nuclei in the same cell, the authors should discuss the possibility that BrdU or EdU treatment has caused irreparable double-stranded breaks in these nuclei.

The authors state that the DNA spreading technique used in this study provides a non-uniform stretching factor but then uses a uniform DNA stretching factor determined previously. Could the authors please elaborate on how it is appropriate to apply this stretching factor when the method used provides non-uniform stretching?

Based on the asynchrony in mitosis, it seems improbable that 100% of mononucleated cells would be replicating over 45 minutes, but this is shown in Figure 5D. Is it possible that this could be the product of nucleotide pre-loading? If so, the authors should discuss this.

Figures

Having all the experimental schematics in Figure 1 before the authors explain what they will be used for seems confusing. It may be more intuitive to include each schematic in the figure/panels of the corresponding assay.

It is difficult to get a sense of the spread of the data in graphs 2a,b,e-h; 3d; 5b,d and 6b,d. The authors should consider presenting some of these data as violin plots, which could help to display this spread better.

There is no explanation for what the dotted circle is in Figures 3a and 4b. The authors should detail this in the figure legends.

Based on their use of cyan and yellow for most of their microscopy, it seems the authors are aware of the difficulties of differentiating red, green, and blue for color-blind readers, and this is very much appreciated. However, it should be noted that the yellow and red used to overlay BrdU and EdU are not discernible for multiple types of color-blindness. Therefore, if color-blind friendliness is important to the authors, they should consider changing the red of EdU to magenta or white.

In the results, the authors imply that there were 100 cells analyzed at each time point in Figure 3, but there seems to be far less than 100 data points for many of the time points in Figure 3D. Therefore, the number of cells should be clarified in the figure legend.

In Figures 4B & D, the second row of graphs seemed confusing. The authors should describe these more thoroughly in the figure legend.

Reviewer #3: Minor points

L62 Although cultured P. knowlesi lines can take 28 hours, this is very likely an artifact of culture (in the same way most cultured Pf are in fact shorter than 48h). In this context it would be worth stating it is ~24 hours.

In a separate but related point, whilst the A1-H.1 has previously been noted to have a lifecycle of ~28hr, it appears to be much longer in these experiments. Can the authors mention this at an appropriate point and suggest a reason? I can’t see anything obvious, but it would be interesting to hear the authors thoughts.

L71 P. cynomologi can also be cultured, albeit in macaque cells. Changing this to state cultured in human red blood cells or among human infective species would be more accurate.

L155 Is budding the correct term here? This suggests progeny growing out from a mother cell?

L164 this is an especially interesting finding. Whilst there is clearly no upper limit to the proportion of dividing nuclei, I am not sure it is quite right to suggest that there are not factors limiting it at the individual cell level. It may be that cells that have 100% replicating nuclei in one round, end up dipping down to 20% due to limitations in the next. It would have been really nice to examine this in the context of limiting nucleotide availability, perhaps limiting hypoxanthine or some other precursors.

L188 and Fig 3 - Figure 3 legend could explain B in more detail. Also the Centrin staining is introduced here but not explained until fig 4 and next results section. Authors could look at order in which these are explained to make its inclusion in Fig 3 clearer. I presume that the Pk 33 hour image is a ring? That is probably not obvious for some readers. Define integrated density in the figure legend for 3D.

Fig 4 – probably artifact of compiling but fig 4 has noticeably lower resolution than other figures.

Fig 5 – Although this experiment is really interesting, it took me a really long time to understand the logic involved (probably my failing!). Could a graphic be added to illustrate it or perhaps further explanation? Perhaps adding this to Fig 1 B and C?

L320 convert this to a full sentence rather than parenthetical?

L443 The Pf and Pk data look quite similar, was the trend towards faster movement in Pk skewed by the two very high speed data points, or did it remain if these were removed? Alter description or not accordingly!

**********

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Reviewer #3: Yes: Robert W. Moon

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PLoS Pathog. 2022 Jun 22;18(6):e1010595. doi: 10.1371/journal.ppat.1010595.r002

Author response to Decision Letter 0

7 Apr 2022

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PLoS Pathog. doi: 10.1371/journal.ppat.1010595.r003

Decision Letter 1

Xin-zhuan Su, Michael J Blackman

5 May 2022

Dear Catherine,

Thank you very much for submitting your manuscript "DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

As you will see, both reviewers appreciate and thank you for the revisions made to this manuscript. However, reviewer #2 remains concerned about your capacity to accurately enumerate individual parasite nuclei based on wide-field epifluorescence microscopic images. Whilst we consider that the overall thrust of the work is compelling, we agree with the reviewer that caution should be exercised in some of the statements made in the manuscript and that some further alterations are warranted. We therefore request that you consider making the following minor changes in a second round of textual revisions (no additional experimental work is required):

1. With regard to supplementary Figure 2 (Figure S2); we agree that the reviewer makes several well-founded criticisms of your interpretation of these images, but we consider that this figure should be retained in the manuscript in order that the data are available to readers. As requested, please provide information on the microscope and deconvolution system used for the confocal microscopic images in Figure S2 panel B.

2. Please consider making the requested changes to Figure 4, Figure 5 and Figure 6 as requested by the reviewer (Fig 4 B and D: use hour post-invasion instead of nuclei number; Fig 4 C and E, Fig 5 E: use replicating DNA instead of replicating nucleus; Figure 6 B, C, E, and F: use DNA masses instead of nuclei). We would suggest that you make it clear in the figure legends, methods or introductory text that the microscopic methodology used inevitably introduces some uncertainly about actual numbers of nuclei in schizonts.

3. Throughout, as recommended by the reviewer, please use the term schizogony (once defined) in place of ‘cell-division’. Use the term ‘nuclear multiplication’ instead of ‘replication’ when referring to karyokinesis, and use ‘DNA replication’ when referring to chromatin synthesis.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

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Michael J Blackman

Associate Editor

PLOS Pathogens

Xin-zhuan Su

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In light of the comments made by the other reviewers and despite my initial assessment I’m happy to trust judgement of the editor about the general suitability of this manuscript for publication in Plos Pathogens. Concerning the scientific integrity of the manuscript all my comments have been sufficiently addressed or at least contextualized appropriately. Hence, I have no further reservations recommending acceptance of this manuscript as is and want to congratulate the authors on their study, which offers an interesting complementary view on the process of schizogony.

Reviewer #2: I want to thank the authors for generating a new set of biological replicates, which strengthens the robustness of the findings. I also want to thank the authors for reaching out to Drs. Ganter and Guizetti best represent their data in the current manuscript and include the most recent published data in the discussion section. However, in the absence of a fluorescent nuclear envelope marker, epifluorescence microscopy does not provide the required resolution to resolve nuclei in multinucleated schizont parasites. In conclusion, I have raised significant comments that need to be addressed in the data representation

in figures 4, 5, and 6 upon acceptance for publication.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: n.a.

Reviewer #2: My last and significant disagreement with the authors remains on the ability to distinguish and resolve a single nucleus in multinucleated schizont parasites without a fluorescent nuclear envelope marker and epifluorescence microscopy.

I am pleased the authors referred to one of my favorite FIBSEM papers by Rudlaff et al. The introduction section explains the limitation of standard fluorescent microscopy.

"The attainable resolution of standard fluorescent microscopy is defined by the Abbe diffraction equation (resolution = λ / (2 * NA), where λ is the wavelength of light used–typically 350–700 nm–and NA is the numerical aperture of the objective–typically <1.5). Practically, this limits the amount of information gained from immunofluorescence microscopy to a resolution of ~200 nm, which does not generally allow separation of structures smaller than this size."

In this paper, the authors mentioned that to generate a robust rendering of a single nucleus, and they called "Nuclear connections for 4n and 2n nuclei were at least 100nm wide through at least 5 Z-sections (100nm deep) to ensure that the observations of connected nuclei were robust" see Data analysis section.

In epifluorescence microscopy, the z-resolution is 2 to 3 times the XY resolution. So therefore, even if the resolution is at 200 nm XY, the z resolution would be between 400 nm to 600 nm, higher than the width of a single nucleus, which ranges from 200 to 350 nm.

Therefore epifluorescence images using DNA staining without a fluorescent nuclear envelope marker can reveal an estimate of DNA masses but not individual nuclei.

Regarding support figure 2, I have multiple concerns, and it supports the fact that only DNA masses and not individual nuclei can be visualized.

For instance, in Panel A, parasite 1: the two -EdU masses with the individual 1C could be either an individual nucleus or a single nucleus at the mitotic spindle phase, parasite 2, the two +EdU top left look very much like one nucleus at the anaphase stage upon nuclear fission, or they could be the individual nucleus. Again the current experimental setting allows to call for DNA masses or replicating DNA and not for individual nucleus.

The authors compared individual z-stack with the maximum projection obtained by wide-field microscopy based on panel B. Confocal microscopy relies on using a pinhole to collect only the fluorescence at the focal point resulting in increasing the resolution. In other terms, with confocal imaging, the fluorescence from objects outside the focal plane is not detected outside of the focal plane, which is not the case in the figure represented in panel B. Could the authors elaborate on what microscope and deconvolution program they use to run their confocal imaging?

In addition, as the author mentions in their manuscript (discussion section): " the centrin foci clearly disappeared in late schizonts after the final round of karyokinesis" therefore, the authors must agree that the third parasite in supplementary figure 2 panel B represents unspecific staining with anti-centrin antibody.

I recommend removing supplementary figure 2 for the final manuscript.

In conclusion, the authors must make the following modifications to their figures and manuscript, ensuring a robust conclusion of their data without changing the main finding of the manuscript.

• Figure 4 Panel B and D: use hour post-invasion instead of nuclei number

• Figure 4 Panel C and E, figure 5 Panel E: use replicating DNA instead of replicating nucleus

• Figure 6 Panel B, C, E, and F: us DNA masses instead of nuclei

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: n.a.

Reviewer #2: The terms cell cycle and schizogony have been used interchangeably in the manuscript (for instance, in the discussion section: lines 370, 371). I would recommend the authors to use the term cell division or stick to schizogony not to confuse a non-malaria biologist. In the same line, it would be best to use the term nuclear multiplication instead of replication when the authors refer to karyokinesis and DNA replication when they refer to chromatin synthesis.

**********

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Reproducibility:

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLoS Pathog. 2022 Jun 22;18(6):e1010595. doi: 10.1371/journal.ppat.1010595.r004

Author response to Decision Letter 1

11 May 2022

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PLoS Pathog. doi: 10.1371/journal.ppat.1010595.r005

Decision Letter 2

Xin-zhuan Su, Michael J Blackman

13 May 2022

Dear Catherine,

We are pleased to inform you that your manuscript 'DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael J Blackman

Associate Editor

PLOS Pathogens

Xin-zhuan Su

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

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***********************************************************

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1010595.r006

Acceptance letter

Xin-zhuan Su, Michael J Blackman

11 Jun 2022

Dear Dr Merrick,

We are delighted to inform you that your manuscript, "DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. DNA replication rates are similar in the newly-generated P. knowlesi TK-expressing line and in the parent line.

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S2 Fig

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S3 Fig. Biological replicates of timecourses shown in Fig 2.

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S4 Fig. Data from Fig 3D (i.e. intensity of EdU staining seen at each timepoint in P. falciparum and P. knowlesi) shown as individual graphs for each category of staining defined in Fig 3B.

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S5 Fig

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S6 Fig. The minimum pulse-labelling period that can be detected via EdU-labelled DNA was tested.

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S7 Fig. Biological replicate of the experiment shown in Fig 4, measuring the length of the first replicative round in P. falciparum and P. knowlesi cells.

Graphs show the percentage of nuclei in 1n cells that labelled with EdU and BrdU (n = 20). Scatter plots display the same data broken down per-cell, with means shown.

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S8 Fig

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Data are also stratified into cells with 2–3, 4–6, or more than 6 nuclear masses, showing that arrested nuclei are still detected in very young 2-3n schizonts.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[ppat.1010595.ref001] 1.Striepen B, Jordan CN, Reiff S, van Dooren GG. Building the perfect parasite: cell division in apicomplexa. PLoS pathogens. 2007;3(6):e78. doi: 10.1371/journal.ppat.0030078 ; PubMed Central PMCID: PMC1904476. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref002] 2.Arnot DE, Ronander E, Bengtsson DC. The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony. International journal for parasitology. 2011;41(1):71–80. doi: 10.1016/j.ijpara.2010.07.012 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref003] 3.Wassmer SC, Taylor TE, Rathod PK, Mishra SK, Mohanty S, Arevalo-Herrera M, et al. Investigating the Pathogenesis of Severe Malaria: A Multidisciplinary and Cross-Geographical Approach. Am J Trop Med Hyg. 2015. doi: 10.4269/ajtmh.14-0841 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref004] 4.Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, et al. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med. 2005;2(8):e204. doi: 10.1371/journal.pmed.0020204 ; PubMed Central PMCID: PMC1188247. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref005] 5.Branco A, Melo-Cristino J. Extreme parasitemia in P falciparum malaria. Blood. 2018;132(8):868. doi: 10.1182/blood-2018-07-861880 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref006] 6.Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419(6906):498–511. doi: 10.1038/nature01097 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref007] 7.Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, et al. Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature. 2008;455(7214):757–63. doi: 10.1038/nature07327 ; PubMed Central PMCID: PMC2651158. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref008] 8.Pain A, Bohme U, Berry AE, Mungall K, Finn RD, Jackson AP, et al. The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature. 2008;455(7214):799–803. doi: 10.1038/nature07306 ; PubMed Central PMCID: PMC2656934. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref009] 9.Stanojcic S, Kuk N, Ullah I, Sterkers Y, Merrick CJ. Single-molecule analysis reveals that DNA replication dynamics vary across the course of schizogony in the malaria parasite Plasmodium falciparum. Sci Rep. 2017;7(1):4003. doi: 10.1038/s41598-017-04407-z ; PubMed Central PMCID: PMC5479783. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref010] 10.Merrick CJ. Transfection with thymidine kinase permits bromodeoxyuridine labelling of DNA replication in the human malaria parasite Plasmodium falciparum. Malar J. 2015;14(490). doi: 10.1186/s12936-015-1014-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref011] 11.Klaus S, Binder P, Kim J, Machado M, Funaya C, Schaaf V, et al. Asynchronous nuclear cycles in multinucleated Plasmodium falciparum facilitate rapid proliferation. Sci Adv. 2022;8(13):eabj5362. doi: 10.1126/sciadv.abj5362 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref012] 12.Reilly HB, Wang H, Steuter JA, Marx AM, Ferdig MT. Quantitative dissection of clone-specific growth rates in cultured malaria parasites. International journal for parasitology. 2007;37(14):1599–607. doi: 10.1016/j.ijpara.2007.05.003 ; PubMed Central PMCID: PMC2268714. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref013] 13.Gerald N, Mahajan B, Kumar S. Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryotic cell. 2011;10(4):474–82. doi: 10.1128/EC.00314-10 ; PubMed Central PMCID: PMC3127633. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref014] 14.Francia ME, Striepen B. Cell division in apicomplexan parasites. Nat Rev Microbiol. 2014;12(2):125–36. doi: 10.1038/nrmicro3184 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref015] 15.Rudlaff RM, Kraemer S, Marshman J, Dvorin JD. Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis. PLoS pathogens. 2020;16(6):e1008587. doi: 10.1371/journal.ppat.1008587 ; PubMed Central PMCID: PMC7302870. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref016] 16.Malinsky J, Koberna K, Stanek D, Masata M, Votruba I, Raska I. The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase. Journal of cell science. 2001;114(Pt 4):747–50. doi: 10.1242/jcs.114.4.747 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref017] 17.Simon CS, Sturmer VS, Guizetti J. How Many Is Enough?—Challenges of Multinucleated Cell Division in Malaria Parasites. Front Cell Infect Microbiol. 2021;11:658616. doi: 10.3389/fcimb.2021.658616 ; PubMed Central PMCID: PMC8137892. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref018] 18.Simon CS, Funaya C, Bauer J, Vobeta Y, Machado M, Penning A, et al. An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites. Life Sci Alliance. 2021;4(11). doi: 10.26508/lsa.202101199 ; PubMed Central PMCID: PMC8473725. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref019] 19.Michalet X, Ekong R, Fougerousse F, Rousseaux S, Schurra C, Hornigold N, et al. Dynamic molecular combing: stretching the whole human genome for high-resolution studies. Science (New York, NY. 1997;277(5331):1518–23. doi: 10.1126/science.277.5331.1518 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref020] 20.Lebofsky R, Bensimon A. Single DNA molecule analysis: applications of molecular combing. Brief Funct Genomic Proteomic. 2003;1(4):385–96. doi: 10.1093/bfgp/1.4.385 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref021] 21.Schwob E, de Renty C, Coulon V, Gostan T, Boyer C, Camet-Gabut L, et al. Use of DNA combing for studying DNA replication in vivo in yeast and mammalian cells. Methods Mol Biol. 2009;521:673–87. doi: 10.1007/978-1-60327-815-7_36 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref022] 22.Parra I, Windle B. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature genetics. 1993;5(1):17–21. doi: 10.1038/ng0993-17 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref023] 23.Jackson DA, Pombo A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. The Journal of cell biology. 1998;140(6):1285–95. doi: 10.1083/jcb.140.6.1285 ; PubMed Central PMCID: PMC2132671. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref024] 24.Garg S, Agarwal S, Dabral S, Kumar N, Sehrawat S, Singh S. Visualization and quantification of Plasmodium falciparum intraerythrocytic merozoites. Syst Synth Biol. 2015;9(Suppl 1):23–6. doi: 10.1007/s11693-015-9167-9 ; PubMed Central PMCID: PMC4688406. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref025] 25.Stewart LB, Diaz-Ingelmo O, Claessens A, Abugri J, Pearson RD, Goncalves S, et al. Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites. Commun Biol. 2020;3(1):624. doi: 10.1038/s42003-020-01349-7 ; PubMed Central PMCID: PMC7595149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref026] 26.Bell DR S.; Patel A.P.; Lee S.H.; Chang Y.T.; Deu E. High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle. BioRxiv. 2021;doi.org/10.1101/2021.03.15.434938 Epub 2021. [Google Scholar]

[ppat.1010595.ref027] 27.Dimitrova DS, Gilbert DM. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat Cell Biol. 2000;2(10):686–94. doi: 10.1038/35036309 ; PubMed Central PMCID: PMC1255923. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref028] 28.Freitas-Junior LH, Bottius E, Pirrit LA, Deitsch KW, Scheidig C, Guinet F, et al. Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature. 2000;407(6807):1018–22. doi: 10.1038/35039531 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref029] 29.Techer H, Koundrioukoff S, Azar D, Wilhelm T, Carignon S, Brison O, et al. Replication dynamics: biases and robustness of DNA fiber analysis. Journal of molecular biology. 2013;425(23):4845–55. doi: 10.1016/j.jmb.2013.03.040 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref030] 30.Mancio-Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, et al. Nutrient sensing modulates malaria parasite virulence. Nature. 2017;547(7662):213–6. doi: 10.1038/nature23009 ; PubMed Central PMCID: PMC5511512. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref031] 31.Trager W, Jensen JB. Human malaria parasites in continuous culture. Science (New York, NY. 1976;193(4254):673–5. doi: 10.1126/science.781840 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref032] 32.Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(2):531–6. doi: 10.1073/pnas.1216457110 ; PubMed Central PMCID: PMC3545754. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref033] 33.Mohring F, Hart MN, Patel A, Baker DA, Moon RW. CRISPR-Cas9 Genome Editing of Plasmodium knowlesi. Bio Protoc. 2020;10(4):e3522. doi: 10.21769/BioProtoc.3522 ; PubMed Central PMCID: PMC7842605. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref034] 34.Donald RG, Zhong T, Wiersma H, Nare B, Yao D, Lee A, et al. Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Molecular and biochemical parasitology. 2006;149(1):86–98. doi: 10.1016/j.molbiopara.2006.05.003 . [DOI] [PubMed] [Google Scholar]

[ppat.1010595.ref035] 35.McDonald J, Merrick CJ. DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi. 2022. Dryad Digital Repository. doi: 10.5061/dryad.ghx3ffbr8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1010595.ref036] 36.Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrobial agents and chemotherapy. 2004;48(5):1803–6. doi: 10.1128/AAC.48.5.1803-1806.2004 ; PubMed Central PMCID: PMC400546. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Jennifer McDonald

Catherine J Merrick

Roles

Abstract

Author summary

Introduction

Results

Timecourse experiments across S-phase in P. falciparum and P. knowlesi

Fig 1. Schematics of experimental setup.

The length of S-phase is proportional to the overall length of erythrocytic schizogony

Fig 2. Length and dynamics of S-phase in P. falciparum and P. knowlesi.

There is no restriction on the number of nuclei that can replicate at once

Patterns of DNA replication within nuclei vary from early to late schizogony

Fig 3. Patterns of DNA replication within nuclei in P. falciparum and P. knowlesi.

Centriolar plaques are dynamic throughout schizogony

Replicative rounds become faster across the course of schizogony

Fig 4. Replicative rounds become faster across the course of schizogony.

Gaps between replicative rounds become longer across the course of schizogony

Fig 5. Gap phases lengthen across the course of schizogony.

Individual nuclei commonly arrest for long periods during schizogony

Fig 6. Long-term arrest of individual nuclei is common during schizogony.

Single-molecule DNA replication dynamics do not differ significantly between P. falciparum and P. knowlesi

Fig 7. Single-molecule DNA replication dynamics in P. falciparum and P. knowlesi.

Discussion

Methods

Parasite culture and transfection for ectopic expression of thymidine kinase

Synchronisation for timecourse experiments

Pulse labelling with modified nucleotides

Immunofluorescence

Data analysis and statistics

DNA fibre spreading

Detection of EdU and BrdU in DNA fibre spreads

Image Acquisition and Processing of DNA fibre data

Availability of raw data

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Xin-zhuan Su

Michael J Blackman

Roles

Author response to Decision Letter 0

Decision Letter 1

Xin-zhuan Su

Michael J Blackman

Roles

Author response to Decision Letter 1

Decision Letter 2

Xin-zhuan Su

Michael J Blackman

Roles

Acceptance letter

Xin-zhuan Su

Michael J Blackman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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