Periodic Reporting for period 4 - PlasmoCycle (DNA dynamics in the unusual cell cycle of the malaria parasite Plasmodium falciparum) Reporting period: 2021-06-01 to 2022-11-30 Summary of the context and overall objectives of the project This project focuses on the malaria parasite Plasmodium. It aims to transform our understanding of the basic biology of Plasmodium, and of how that biology affects virulence. This is a vitally important topic for research because malaria imposes a huge burden of disease on human populations: ~0.4 million deaths and >200 million clinical cases per year. The malaria parasite is an unusual, early-diverging protozoan lifeform. Its basic biology is very different from that of its human host. In particular, it pursues a complex lifecycle with modes of cell division that differ at different lifecycle stages. Remarkably little is known about Plasmodium cell cycles, despite a wealth of knowledge on the subject in human cells. This project will reveal, with unprecedented resolution, how DNA replication is organised in Plasmodium and how it can be affected by changing conditions in the human host and exposure to antimalarial drugs.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 from human cells to yeast cells. In schizogony a single parasite first generates many nuclei via independent, asynchronous rounds of genome replication, prior to cytokinesis which is the physical division of the cell. This takes a period of ~24 hours when the parasite is inside human red blood cells. However, the genome can also be copied extremely rapidly during the sexual cycle, which occurs in the malaria-transmitting mosquito. Here 8 male gametes are produced from a single ‘gametocyte’ in less than 10 minutes, necessitating extraordinarily rapid DNA synthesis. Thus, schizogony challenges some basic paradigms about DNA replication control, while gametogenesis demands a speed of DNA replication and cell division that is unprecedented in eukaryotic gametogenesis.This project is elucidating the spatio-temporal dynamics of DNA replication in these contrasting cell cycles. To do this, I have developed a method for labelling nascent DNA replication, which was not hitherto possible in Plasmodium. This method now permits: a) a detailed characterisation, at the whole-cell level, of the asynchronous genome replication that occurs in schizogony and gametogenesis; b) a study of replication-origin spacing and DNA synthesis speed at single-molecule resolution on DNA fibres, comparing these parameters in schizogony and gametogenesis; c) mapping of sequences with replication origin activity in the Plasmodium genome; d) investigation of cell-cycle checkpoints and replicative responses to the changing environment in the human host and to antimalarial drugs. These are crucial issues for understanding malaria parasite virulence and drug-resistance. The work will ultimately inform vital new research into transmission-blocking interventions for malaria. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Substantial progress has been made within the first 2.5 years on Aims 1-3 of this 5-year, 4-Aim project.Aim 1: detailed characterisation, at the whole-cell level, of the asynchronous genome replication that occurs in schizogony and gametogenesis.Timecourses have been conducted to quantify the number and type of replicating nuclei, together with other cell-biological features, in P. falciparum parasites across the course of both erythrocytic schizogony and gametogenesis. Triplicate datasets have been generated for gametogenesis and the results were presented on a poster by PDRA Holly Matthews at the annual Molecular Parasitology Meeting (MPM) in the USA in Sept 2018 (~1.3 years into the project). Data gathering remains underway for the process of schizogony, and this investigation has also been expanded to include comparative work in P. knowlesi, another malaria parasite with a cell cycle twice as fast as that of P. falciparum. The main results of the gametogenesis study include a) the observation that nuclei do not condense or visibly divide after each replicative round, but only at the end of replication, and b) some parasites can exflagellate without undergoing any DNA replication, suggesting that the processes of replication and cytokinesis – normally sequential – are unlinked and that cell cycle checkpoints are absent in gametogenesis. The main results of the schizogony study include a) the first observation in Plasmodium of subnuclear ‘replication factories’ where nascent DNA synthesis is concentrated together with ‘ORC’ origin-of-replication protein complexes, and b) successive rounds of nuclear replication are not only asynchronous but also non-continuous: at any time only ~50-60% of nuclei in a schizont are actively replicating, until the final replicative round, when all nuclei appear to replicate together, much more slowly than in previous rounds.These studies will form the basis of two significant papers to be written within 2020: one on the dynamics of gametogenesis in P. falciparum and one on the dynamics of schizogony in P. falciparum as compared with P. knowlesi.Aim 2: to study replication-origin spacing and DNA synthesis speed at single-molecule resolution on DNA fibres, comparing these parameters in schizogony and gametogenesis.During the process of negotiation and staff-hiring for this grant in 2017, a first tranche of work on this Aim was pursued and published ('Single-molecule Analysis Reveals that DNA Replication Dynamics Vary Across the Course of Schizogony in the Malaria Parasite Plasmodium falciparum.' Stanojcic, S., Kuk, N., Ullah, I., Sterkers, Y., Merrick, C.J. Scientific Reports 7:4003 (2017)). Thus, we have already established parameters for replication-origin spacing and DNA synthesis speed during schizogony. We have since worked to establish the same parameters for the much more technically-challenging and highly accelerated process of gametogenesis. A gametocyte-producing strain of P. falciparum was genetically engineered such that nascent DNA replication could be labelled, gametocytes were generated from this strain, stimulated to replicate, and pulse-labelled DNA fibres were produced. The main result of this work thus far has been the clear observation that gametocyte replication is indeed extremely fast: after just 30s of pulse-labelling, it is impossible to measure discrete tracks of nascent DNA replication because strands of DNA several 100kb long are completely replicated within 30s. (By contrast, discrete tracks are still visible after 20mins of nascent DNA labelling during schizogony.) We are now working on a new single-molecule technique (see Aim 3) to determine the replication-origin spacing that underlies this rapid replication, because the time/space resolution obtainable on DNA fibres appears to be limiting. These data will contribute, within 2020, to the manuscript outlined above on gametogenesis in P. falciparum.Aim 3: map sequences with replication origin activity in the Plasmodium genome.Substantial technical development has gone into Aim 3 and results are now finally beginning to emerge. Replication origins were to be mapped by two complementary techniques: ChiP for an ORC protein (marking all putative origins) and pulldown of BrdU-labelled nascent DNA fragments (denoting only active origins). For the first approach, a strain has been generated in which an endogenous ORC gene is tagged with a 3xHA tag; this strain has been characterised and an initial ChIP experiment has been conducted. Troubleshooting is presently underway to improve the specificity of this ChIP. For the second approach, technical challenges have led to a re-strategizing and the adoption of a new experimental approach, which is now beginning to produce exciting preliminary results. A publication is therefore anticipated by the end of 2020.Aim 4: investigate cell-cycle checkpoints and replicative responses to the changing environment in the human host and to antimalarial drugs.This final aim of the grant has not yet been substantially addressed within the first 2.5 years. After completing the work outlined above on Aims 1-3 within 2020, the two PDRAs will turn their attention to Aim 4 during the final ~1.5 years of the grant. In addition, I have just recruited two new graduate students (PhD and MPhil) who will work on a project studying cell-cycle checkpoints, which is highly relevant to this Aim. These students are not fully funded by this grant, but their lab consumables will cross-subsidised and their work will inform the overall direction of Aim 4. In addition to the 3 manuscripts in preparation that are detailed above, an invited Review Article was published in 2018, reviewing the state of the Plasmodium cell cycle field. ('Checks and Balances? DNA replication and the cell cycle in Plasmodium.' Matthews, H., Duffy C.W. Merrick, C.J. Parasites & Vectors (2018)) Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The majority of these results do advance the state of the art regarding our knowledge of Plasmodium DNA replication. In Aim 1, the time-resolution with which DNA replication can be followed through schizogony and gametogenesis via BrdU labelling is unprecedented in the literature thus far. (In fact, ongoing work by a colleague using continuous live-cell imaging, as opposed to a timepoint-series of fixed cells, improves this time-resolution still further, but at the expense of the spatial resolution that we can obtain in fixed cells. Work by our two groups is therefore highly complementary.) Overall, the state of the art in this area is clearly fast advancing.In Aim 2, DNA replication in Plasmodium has never previously been examined at single-molecule resolution, so all the data thus generated are novel. In particular, the process of replication in gametocytes has never been accessible at this resolution. Understanding this process, and the associated presence or absence of cell cycle checkpoints, is likely to be impactful because this fast, poorly-regulated process represents a potential ‘Achilles heel’ for the parasite – vulnerable to replication-damaging interventions such as drugs, and crucial for the transmission of malaria.In Aim 3, replication origins have never previously been mapped in Plasmodium, nor in any organism with such a highly A/T-biased genome. Therefore, all data generated will advance the state of the art. Furthermore, Plasmodium offers a unique opportunity to compare origin determination in two genomes with similar cell cycles but vastly different A/T contents (P. falciparum and P. knowlesi) – this is expected to yield fundamental insights about replication origin determination. Wider societal impacts have not occurred thus far, but this is as expected from a basic-biology project at the 2.5 year mark, particularly when working with a pathogen in which molecular genetics is notoriously slow and meaningful translational work tends to be similarly slow.