Asynchronous nuclear cycles in multinucleated Plasmodium falciparum enable rapid proliferation

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

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

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