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Asynchronous nuclear cycles in multinucleated Plasmodium falciparum enable rapid proliferation

Severina Klaus, Patrick Binder, Juyeop Kim, Marta Machado, Charlotta Funaya, Violetta Schaaf, Darius Klaschka, Aiste Kudulyte, Marek Cyrklaff, Vibor Laketa, Thomas Höfer, Julien Guizetti, Nils B. Becker, Friedrich Frischknecht, Ulrich S. Schwarz, Markus Ganter

Preprint posted on April 15, 2021 https://www.biorxiv.org/content/10.1101/2021.04.15.440016v1

How Plasmodium parasites multiply into multinucleated cells

Selected by Joao Mello-Vieira

Categories: cell biology, microbiology

Background

Plasmodium parasites are the causative agents of the tropical disease malaria. This disease caused over 200 million cases in 2019 and was responsible for 400 thousand deaths. During their complex life cycle, parasites only cause disease when they infect erythrocytes, in what is called the “blood stage of infection”. While infecting red blood cells, each Plasmodium falciparum parasite gives rise to an average of 20 new daughter parasites in a process that takes approximately 48 hours.

Cellular multiplication in these parasites is a complex process termed schizogony; they perform multiple rounds of DNA replication and nuclear division with just one cytokinesis event. This means that at the latter stages of an erythrocyte infection, parasites can have several nuclei sharing the same cytoplasm, creating a multinucleated cell. Besides, Plasmodium nuclei replicate asynchronously, with each nucleus behaving almost like an independent unit. Nevertheless, Plasmodium parasites achieve a high replication output with approximately 4 to 5 rounds of replication in 48 hours. In this preprint, Severina Klaus and colleagues try to untangle this complex phenomenon by tracking individual nuclei before, during and after they replicate.

To study the replication dynamics of the Plasmodium falciparum parasite, the authors created a fluorescent reporter protein, PCNA1::GFP, whose presence in the nucleus of the parasites coincides with the increase of DNA content. This means that when the authors observed this protein in the nucleus of a parasite, that particular nucleus would replicate its DNA. Using this reporter protein as a proxy for DNA duplication, the authors could follow DNA replication and mitosis in Plasmodium parasites with a fluorescence microscope.

 

Plasmodium falciparum-infected erythrocyte. This parasite expresses PCNA1-GFP (in green), which localizes to the nucleus that is replicating its DNA. DNA is labelled in pink, scale bar is 1 μm.

 

Key results

  • Using parasites that express the reporter protein PCNA1::GFP, the authors observed that DNA replication is asynchronous in Plasmodium falciparum Each nucleus divides independently of neighbouring nuclei. In parasites with two nuclei, approximately 40% of the mitotic events, there is only one nucleus duplicating its DNA at a given moment. In only 20% of the mitotic events the authors see complete overlap between two nuclei replicating its DNA. In the remaining 40%, there is a partial overlap between mitosis.
  • Using these reporter parasites, the authors could determine how much time it took for a nucleus to duplicate its DNA (S-phase), how long it took for each nuclear division (S-phase to division) and how long it took for a new daughter nucleus to start duplicating its DNA (after division to S-phase). It seems that the first nuclear cycle is the longest, not only during the S phase, but also in the time it takes for the two nuclei to divide.
  • Lastly, the authors compare two conceptual models to better understand the dynamics of nuclear division: a model where parasites had a fixed interval of time to divide (Time model) and a model where parasites divide until a given number of nuclei is reached (Counter model). With all the measurements obtained with the reporter parasites, the authors observed that the total time that parasites took to replicate was correlated with the duration of the first mitotic cycle. This means that parasites did not stop replicating after a fixed interval of time. This suggested to the authors, that the Counter model was the model that better explains the dynamics of Plasmodium parasites multiplication.

 

Importance

Schizogony, the multiplication process of Plasmodium parasites, is a complex process. In this preprint, the authors confirmed that Plasmodium parasite DNA duplication in sister nuclei during the blood stage of infection is indeed asynchronous, as is its nuclear division. Given that each parasite gives rise to approximately 20 new daughter cells in 48 hours, it is plausible to think that these parasites evolved this asynchronicity to balance limited resources with rapid proliferation. Moreover, when nuclei divide their DNA at the same time, the DNA replication speed decreases.

Moreover, the duration of each replication round is variable. For instance, the first replication event (from 1 nucleus to 2) is the one that takes the longest, taking 120 minutes on average. Conversely, the second replication round (2 nuclei to 4) is the fastest, taking on average 80 minutes. Subsequent rounds thereafter seem to be slower than the second round. Using a mathematical model to better understand the data, the authors observed that slowing down the replications round from the second event onwards by 17% fitted the experimental data better. This can imply that the existence of a limiting factor that is consumed by the parasite. This factor can be a protein (DNA polymerase, for example), a cofactor (nucleotides) or even nutrients (glucose).

Finally, a study like this also gives clues to the shortcomings of our concepts. For example, one typical measurement of Plasmodium fitness is the number of nuclei the parasite creates 48h after it infects an erythrocyte, the time for one infection cycle. However, if the Counter model of the authors is correct, the 48h mark does not mean the end of the infection cycle. The parasite might keep replicating until a satisfactory number of new daughter cells is reached.

In summary, the authors created a Plasmodium falciparum parasite line that can be used to visualize DNA replication and nuclear division. This parasite line is useful as a tool to study the mode of action of anti-malarial compounds and also to advance our understanding of Plasmodium parasite biology.

Tags: dna replication, microscopy, mitosis

Posted on: 24th May 2021

doi: https://doi.org/10.1242/prelights.29113

Read preprint (2 votes)




Author's response

Severina Klaus and Markus Ganter shared

João Mello-Vieira (JMV): You mentioned in the introduction that the last replication cycle of Plasmodium parasites is synchronized. How do you incorporate that into your model? For example, do you think that parasites “count” to 10 and then replicate all 10 nuclei into 20?

Severia Klaus and Markus Ganter (SK/MG): Our data indeed supports that stopping nuclear multiplication is based on a counter mechanism. The prevailing view is that there is a final synchronous nuclear cycle (including S-phase and mitosis of course), which is coordinated with cellularization. Our data, however, supports the notion that S-phases occur asynchronously until DNA replication ceases. We found no evidence for a final synchronous round of S-phase in all nuclei. It appears that the last S-phase is not directly followed by mitosis and that nuclei remain in diploid state until the final relatively synchronous mitosis occurs that coincides with daughter cell formation. Thus, our mathematical model assumes that at some point during schizogony S-phases are no longer initiated but the nuclei actively undergoing S-phase at this point will finish.

 

JMV: What do you think is the limiting factor that slows down replication?

SK/MG: Good question, we hope to find out. Generally speaking, it may be extrinsic factors and for example low glucose levels have been shown to lead to a reduced number of daughter cells (Mancio-Silva et al., 2017 (PMID:28678779). However, even in cell culture where all parasites reside in the same environment, they produce varying numbers of daughter cells. This suggests a possible additional role of intrinsic factors that may by rate limiting, such as proteins with a critical role for S-phase.

 

JMV: For male gametocytogenesis, Plasmodium falciparum parasites achieve 3 rounds of replication very rapidly. In this case, is it possible to achieve this genomic output with asynchronous replication with progressive slowing down after each round? Or do you think this might be due to different conditions (nutrients, protein abundance) that lead to a faster mitotic output?

SK/MG: It is very difficult to extrapolate our findings to other life cycle stages, especially the activated male gametocyte. The cell cycle events that occur in the male gametocyte are very different from those that occur during schizogony. Also, the time scales vary dramatically. Raabe et al., 2009 (PMID:19712704) quantified the DNA content of a population of activated male gametocytes over time and found a stepwise increase. Perhaps this indicates a more synchronous progression. To our knowledge the dynamics of the individual S-phases in activated male gametocytes have not yet been determined.

 

JMV: What to do you think is counting the number of nuclei? Do you think that it’s the same protein complex that operates in each stage of the parasite life cycle, like in ookinetes or liver stage schizonts?

SK/MG: The concept of a counter mechanism is part of a larger fundamental question of how cells balance their growth with their division to achieve size homeostasis. Counter (or sizer) mechanisms can be quite diverse (Facchetti et al., 2017 PMID:32984663). For human cells and budding yeast, it was shown that cell growth dilutes an inhibitor and under a certain threshold, cell division is triggered (Schmoller et al., 2015, PMID:26390151; Zatulovskiy et al., 2020, PMID:32703881). In fission yeast, an activator accumulates over time and triggers cell division (Pan et al 2014, PMID:24642412). It remains to be seen if and how these concepts can be transferred to the counter mechanism that stops nuclear multiplication in the Plasmodium blood stage. Currently, we know too little about the proliferation of the other life cycle stages to make any predictions on the mechanism that controls size homeostasis.

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