Periodic Reporting for period 5 - PlasmoCycle (DNA dynamics in the unusual cell cycle of the malaria parasite Plasmodium falciparum)
Berichtszeitraum: 2022-12-01 bis 2023-11-30
This project focussed on the malaria parasite Plasmodium. It aimed to transform our understanding of the parasite's basic biology, and of how that biology affects virulence. This is a vital topic for research because malaria imposes a huge burden of disease on humanity: about half a million deaths & >200 million clinical cases per year.
The malaria parasite is an unusual, early-diverging protozoan. Its basic biology is very different from that of its human host. In particular, it has a complex lifecycle with modes of cell division that differ at different lifecycle stages. Remarkably little was known about this, despite a wealth of knowledge in human cells. Our project revealed, with unprecedented resolution, how DNA replication is organised in Plasmodium.
Malaria parasites replicate inside the cells of their human host via ‘schizogony’, which is fundamentally different from conventional binary fission (the replication mode used by most cells). A single parasite first generates many nuclei via independent, asynchronous rounds of genome replication prior to cytokinesis – the physical division of the cell. Replication takes a period of tens of hours when the parasite is inside human red blood cells, but the genome can also be copied extremely rapidly during the sexual cycle inside the malaria-transmitting mosquito. Here, 8 male gametes are produced from a single ‘gametocyte’ in less than 10 minutes, via extraordinarily rapid DNA synthesis. Thus, schizogony challenges basic paradigms about DNA replication control, while gametogenesis demands a speed of DNA replication unprecedented in eukaryotic gametogenesis.
This project elucidated the spatio-temporal dynamics of DNA replication in these contrasting cell cycles. We studied the control of asynchronous genome replication in schizogony and also in gametogenesis. We mapped replication origin activity across the Plasmodium genome, and determined for the first time the parameters of replication-origin spacing and DNA synthesis speed at single-molecule resolution on DNA fibres. We characterised cell-cycle checkpoints in Plasmodium and how replication responds to changing environments in the human host, and to antimalarial drugs. These are crucial issues for understanding malaria parasite virulence and drug-resistance. Our work has already revealed new details about how parasite replication responds to antimalarial drugs, potentially guiding development of better synergistic treatments.
The malaria parasite is an unusual, early-diverging protozoan. Its basic biology is very different from that of its human host. In particular, it has a complex lifecycle with modes of cell division that differ at different lifecycle stages. Remarkably little was known about this, despite a wealth of knowledge in human cells. Our project revealed, with unprecedented resolution, how DNA replication is organised in Plasmodium.
Malaria parasites replicate inside the cells of their human host via ‘schizogony’, which is fundamentally different from conventional binary fission (the replication mode used by most cells). A single parasite first generates many nuclei via independent, asynchronous rounds of genome replication prior to cytokinesis – the physical division of the cell. Replication takes a period of tens of hours when the parasite is inside human red blood cells, but the genome can also be copied extremely rapidly during the sexual cycle inside the malaria-transmitting mosquito. Here, 8 male gametes are produced from a single ‘gametocyte’ in less than 10 minutes, via extraordinarily rapid DNA synthesis. Thus, schizogony challenges basic paradigms about DNA replication control, while gametogenesis demands a speed of DNA replication unprecedented in eukaryotic gametogenesis.
This project elucidated the spatio-temporal dynamics of DNA replication in these contrasting cell cycles. We studied the control of asynchronous genome replication in schizogony and also in gametogenesis. We mapped replication origin activity across the Plasmodium genome, and determined for the first time the parameters of replication-origin spacing and DNA synthesis speed at single-molecule resolution on DNA fibres. We characterised cell-cycle checkpoints in Plasmodium and how replication responds to changing environments in the human host, and to antimalarial drugs. These are crucial issues for understanding malaria parasite virulence and drug-resistance. Our work has already revealed new details about how parasite replication responds to antimalarial drugs, potentially guiding development of better synergistic treatments.
In the course of this project, 4 related Aims were addressed.
Aim 1 was a detailed characterisation of the asynchronous genome replication that occurs in schizogony and gametogenesis. This was achieved, resulting in two publications in 2022 (McDonald & Merrick, Plos Pathogens; Matthews et al. Cell Microbiol) and presentations at international conferences by Dr Matthews (MPM 2018) & Dr McDonald (BioMalPar 2022). In schizogony, we found that replication dynamics differed significantly between two Plasmodium species, P. knowlesi & P. falciparum, and there was extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some nuclei arresting for many hours while adjacent nuclei continued to replicate. In gametogenesis we saw that nuclei do not condense or divide after each replicative round; that the processes of replication and cytokinesis – normally sequential – are unlinked; and that cell cycle checkpoints are absent.
Aims 2 & 3 concerned DNA replication at single-molecule resolution. Replication origin spacing and DNA synthesis speed were measured, and we mapped origin activity across the Plasmodium genome. This produced the first map of replication origin activity in this A/T-rich genome, and discerned associated features, such as low transcriptional activity. We attempted the same measurements in the faster process of gametogenesis, but this proved technically challenging and is still ongoing. We published our results in Scientific Reports (2017), and Nucleic Acids Research (2023), plus a conference presentation by Dr Totanes in 2022. We also extended our techniques to make a comparison with the very different P. knowlesi genome: this work is being prepared for publication.
Aim 4 was to investigate cell-cycle checkpoints and replicative responses to the changing environment in the human host and to antimalarial drugs. Four papers around this topic are in preparation. We have determined that checkpoint-like activity occurs in Plasmodium schizogony, and have pursued a hypothesis about unconventional checkpoint-mediating protein(s). We have characterised the replicative impact of glucose & isoleucine starvation – nutritional conditions that can occur naturally in malaria patients – and likewise of the impact of DNA-damaging antimalarial drugs. When published, our findings could have real impacts in guiding the development of new antimalarial drugs.
Aim 1 was a detailed characterisation of the asynchronous genome replication that occurs in schizogony and gametogenesis. This was achieved, resulting in two publications in 2022 (McDonald & Merrick, Plos Pathogens; Matthews et al. Cell Microbiol) and presentations at international conferences by Dr Matthews (MPM 2018) & Dr McDonald (BioMalPar 2022). In schizogony, we found that replication dynamics differed significantly between two Plasmodium species, P. knowlesi & P. falciparum, and there was extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some nuclei arresting for many hours while adjacent nuclei continued to replicate. In gametogenesis we saw that nuclei do not condense or divide after each replicative round; that the processes of replication and cytokinesis – normally sequential – are unlinked; and that cell cycle checkpoints are absent.
Aims 2 & 3 concerned DNA replication at single-molecule resolution. Replication origin spacing and DNA synthesis speed were measured, and we mapped origin activity across the Plasmodium genome. This produced the first map of replication origin activity in this A/T-rich genome, and discerned associated features, such as low transcriptional activity. We attempted the same measurements in the faster process of gametogenesis, but this proved technically challenging and is still ongoing. We published our results in Scientific Reports (2017), and Nucleic Acids Research (2023), plus a conference presentation by Dr Totanes in 2022. We also extended our techniques to make a comparison with the very different P. knowlesi genome: this work is being prepared for publication.
Aim 4 was to investigate cell-cycle checkpoints and replicative responses to the changing environment in the human host and to antimalarial drugs. Four papers around this topic are in preparation. We have determined that checkpoint-like activity occurs in Plasmodium schizogony, and have pursued a hypothesis about unconventional checkpoint-mediating protein(s). We have characterised the replicative impact of glucose & isoleucine starvation – nutritional conditions that can occur naturally in malaria patients – and likewise of the impact of DNA-damaging antimalarial drugs. When published, our findings could have real impacts in guiding the development of new antimalarial drugs.
Collectively, we have advanced the state of the art in our knowledge of Plasmodium DNA replication. We have produced and published a suite of high-resolution tools that can now be used for mapping and quantifying replication under any condition, such as antimalarial drug pressure. We have also produced a highly-cited review, a well-received hypothesis article, and a special journal collection with associated editorial, all promoting this fast-growing field of research.
In Aim 1, we followed DNA replication through schizogony and gametogenesis with a time-resolution that was completely unprecedented in the literature. A colleague has since developed continuous live-cell imaging (as opposed to timepoint-series of fixed cells), improving time-resolution still further, albeit at the expense of spatial resolution. We are now working together to exploit these complementary techniques. Others in the field are exploiting high-resolution microscopy such as FIB-SEM. Overall, the state of the art in this area is advancing rapidly. In particular, our collective understanding of the fast, poorly-regulated process of gametogenesis has greatly improved. This represents a potential ‘Achilles heel’ for the parasite – vulnerable to replication-damaging interventions such as drugs, and crucial for the transmission of malaria.
In Aims 2 and 3, DNA replication had never previously been examined at single-molecule resolution in Plasmodium, nor in any organism with such a highly A/T-biased genome. All our data on this topic are novel and, thus far, unique. The technique we developed (single-molecule replication mapping via nanopore sequencing) laid the groundwork in understanding the landscape of replication origins & replication fork speed in Plasmodium. It has great potential for high-resolution studies of the acute effects of antimalarial drug treatment, which may stall replication forks. It also has wider potential in fields such as cancer research. In terms of basic biology, Plasmodium also offered us a unique opportunity to compare replication origin activity in two genomes with similar cell cycles but vastly different A/T contents (P. falciparum & P. knowlesi) – this is yielding fundamental insights about replication origin determination in ongoing work within Aim 3.
Wider societal impacts have not occurred thus far, as expected from a project focussed on basic-biology and technique development, but there are real prospects for future impact. These are in guiding development of synergistic antimalarial drugs and/or anti-transmission drugs.
In Aim 1, we followed DNA replication through schizogony and gametogenesis with a time-resolution that was completely unprecedented in the literature. A colleague has since developed continuous live-cell imaging (as opposed to timepoint-series of fixed cells), improving time-resolution still further, albeit at the expense of spatial resolution. We are now working together to exploit these complementary techniques. Others in the field are exploiting high-resolution microscopy such as FIB-SEM. Overall, the state of the art in this area is advancing rapidly. In particular, our collective understanding of the fast, poorly-regulated process of gametogenesis has greatly improved. This represents a potential ‘Achilles heel’ for the parasite – vulnerable to replication-damaging interventions such as drugs, and crucial for the transmission of malaria.
In Aims 2 and 3, DNA replication had never previously been examined at single-molecule resolution in Plasmodium, nor in any organism with such a highly A/T-biased genome. All our data on this topic are novel and, thus far, unique. The technique we developed (single-molecule replication mapping via nanopore sequencing) laid the groundwork in understanding the landscape of replication origins & replication fork speed in Plasmodium. It has great potential for high-resolution studies of the acute effects of antimalarial drug treatment, which may stall replication forks. It also has wider potential in fields such as cancer research. In terms of basic biology, Plasmodium also offered us a unique opportunity to compare replication origin activity in two genomes with similar cell cycles but vastly different A/T contents (P. falciparum & P. knowlesi) – this is yielding fundamental insights about replication origin determination in ongoing work within Aim 3.
Wider societal impacts have not occurred thus far, as expected from a project focussed on basic-biology and technique development, but there are real prospects for future impact. These are in guiding development of synergistic antimalarial drugs and/or anti-transmission drugs.