Biology subjects

Merrick, C. J.

Publications and source records attributed to Merrick, C. J..

15 recordsLinked to original sources

bioRxiv2026

Nanopore sequencing reveals the DNA replication timing programme of the malaria parasite Plasmodium falciparum

The DNA replication timing programme, defined as the temporal order in which genomic loci replicate, is of fundamental importance to maintaining genome stability. In human cells, replication timing is routinely measured using Repli-seq which reveals that the human genome is divided into replication timing domains that replicate in early or late S-phase. These timing domains are associated with gene expression, chromatin compaction, and three-dimensional genome organisation. In particular, late-replicating domains can be hotspots of genomic instability under replication stress. Because of the parasite's unusual cell cycle, Repli-seq cannot be used in the malaria parasite Plasmodium falciparum, leaving the open question as to whether its genome is divided into replication timing domains and how this might impact evolution of the parasite. Here, we leverage the power of Oxford Nanopore Technologies long-read DNA sequencing together with DNAscent to determine, for the first time, the replication timing programme of P. falciparum. We show how it relates to elevated rates of DNA breaking and replication fork stalling, and predict that these stalls are predominantly transient.

genomics↗

bioRxiv2026

L-lactate is a regulator of gametocytogenesis in Plasmodium falciparum

Epigenetic processes play important roles in the biology of the malaria parasite Plasmodium falciparum. We recently showed that Plasmodium histones are not only acetylated and methylated but also lactylated. This new epigenetic mark could form a particularly important axis of host-parasite signalling because severe malaria is often characterised by hyperlactataemia. In the human host this can cause respiratory distress, which is potentially fatal. For the parasite, it could be advantageous to sense this state of pathology and respond by modulating virulence. Virulence processes known to be under epigenetic control include antigenic variation, invasion switching and conversion to sexual gametocytes, i.e. gametocytogenesis. Here, we investigated the influence of lactate on gametocytogenesis. Hyperlactataemia in malaria patients is defined as [≥] 5mM blood lactate and we confirmed that in laboratory culture, adding 5mM L-lactate was sufficient to boost gametocytogenesis. Expression of the GDV-1 gene increased within one cell cycle of asexual parasites being exposed to L-lactate, suggesting that the canonical epigenetic switch for gametocytogenesis was involved: GDV-1 regulates epigenetic de-repression of the gene encoding the master transcription factor for gametocytogenesis, AP2-G. Indeed, parasite histones became rapidly lactylated after L-lactate exposure and chromatin profiling by CUT&Tag detected inducible lactylation of chromatin specifically upstream of the AP2-G gene, and also of the antisense RNA that regulates GDV-1. Thus L-lactate joins S-adenosylmethionine as a metabolite that can epigenetically regulate gametocytogenesis in P. falciparum.

microbiology↗

bioRxiv2026

Lactylated histones mark virulence gene families in the malaria parasite Plasmodium falciparum

Epigenetic pathways have many important roles controlling virulence in human malaria parasites. Histone acetylation and methylation have been closely studied in this context but the novel epigenetic mark of lactylation has not yet been examined. Here, for the first time, we profiled lactyl-histone marks across the P. falciparum genome and found them strongly enriched at virulence genes, including genes involved in cytoadhesion and other host-cell remodelling functions. Many genes were dynamically and inducibly lactylated across the cell cycle. Thus, P. falciparum could use histone lactylation to control its virulence pathways in response to the prevailing metabolic environment in its host. We extended our chromatin profiling to parasites isolated directly from human patients, showing that here too, virulence gene families were strongly lactylated. This represents the first comprehensive profiling of P. falciparum chromatin from parasites in sub-millilitre blood samples, opening up exciting new avenues to study parasite chromatin across human disease states.

microbiology↗

bioRxiv2025

Cell cycle checkpoint activity in the malaria parasite Plasmodium falciparum

Plasmodium spp. have different modes of cell division from most eukaryotes. Little is known about how these are controlled and cell-cycle checkpoints are particularly poorly characterised. However, parasites can arrest their cell cycle when treated with the frontline antimalarial drug artemisinin, and artemisinin-resistant parasites can modulate their cell-cycle progression, so it is important to understand these aspects of Plasmodium biology. Here, we show that P. falciparum displays hallmarks of an intra-S-phase checkpoint when exposed to DNA damage, including acute reduction of DNA replication and phosphorylation of a putative damage-marker histone. Compounds that inhibit human checkpoint kinases can inhibit this arrest of DNA replication, and synergise with DNA damage in parasite killing. This suggests the existence of checkpoint kinase activity in P. falciparum, yet these kinases have no clear homologues in Plasmodium genomes. Their closest homologues are the phosphatidylinositol lipid kinases. We hypothesise that phosphatidylinositol 3-kinase - which is reportedly up-regulated in artemisinin-resistant parasites - may moonlight in this role, and we characterise this essential kinase for the first time via expansion microscopy. Finally, we show that the cryptic checkpoint-kinase activity may also regulate the ring-stage survival phenotype after artemisinin damage, which resembles a G1/S checkpoint. Hence we suggest that checkpoint kinase inhibitors are candidates for synergy with artemisinin. ImportanceMalaria parasites infect red blood cells, wherein they replicate to produce many new parasites. This is unusual because most cells replicate simply by copying their genome and splitting in half (called binary fission) but malaria parasites make [~]20 genome copies, then partition them simultaneously into 20 new cells (called schizogony). Here we studied how schizogony is controlled: in particular, are there checkpoints? Checkpoints pause the cycle for repair if the genome is damaged. We found that DNA damage did cause checkpoint hallmarks, yet key proteins that enforce this in other cells are absent in malaria parasites. Furthermore, this checkpoint activity may be involved in the response to an antimalarial drug, when parasites pause their cycle before active replication begins. This implies that inhibiting the checkpoint could exacerbate parasite killing by such drugs. Cancer therapies often work like this - by damaging DNA and also preventing the cancer cells from repairing it.

microbiology↗

bioRxiv2024

DNA replication dynamics are associated with genome composition in Plasmodium species

Plasmodium species have variable genome compositions: many have an A/T-content of at least 80% while others are similar in composition to human cells. Here, we made a direct comparison of DNA replication dynamics in two Plasmodium species whose genomes differ by [~]20% A/T-content. This yielded fundamental insights into how DNA composition may affect replication. The highly A/T-biased genome of P. falciparum showed unusual replication dynamics that were not observed in the more balanced P. knowlesi - which had dynamics more like those of human cell lines. We observed that replication forks moved 50% slower in P. falciparum than in P. knowlesi. In P. falciparum, replication forks slowed down over the course of S-phase whereas in P. knowlesi, fork speed increased as in human cells. Furthermore, in both P. knowlesi and human cells, replication forks were strikingly slowed by sequences of particularly high A/T-bias, but in P. falciparum, although replication forks were inherently slow, they were not particularly slow in such biased sequences. Thus, the replisome of P. falciparum may have evolved alongside its extremely biased genome, making it unusually robust to sequence bias. Since several antimalarial drugs act to stall DNA replication, this study may have implications for the effectiveness of, and development of, antimalarial therapies.

microbiology↗

bioRxiv2024

Characterisation of cell cycle checkpoint kinases in Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite in the apicomplexan phylum. Apicomplexan parasites replicate using a variety of non-canonical cell division modes, distinct from binary fission, whose molecular regulation is incompletely understood. T. gondii replicates by endodyogeny in its intermediate hosts, and by endopolygeny in its definitive host. To improve our understanding of how these unusual, flexible cell division modes are regulated, we characterised the T. gondii homologues of the cell-cycle checkpoint kinases ATM and ATR. These phosphoinositol-3-kinase-like kinases are entirely absent in some related parasites including Plasmodium; in T. gondii they are present but their putative checkpoint roles were previously uncharacterised. Both TgATM and TgATR were found to be dispersed throughout the parasite, rather than restricted to the nucleus, and they did not detectably relocate to the nucleus after DNA damage. Nevertheless, they were both required for checkpoint responses to DNA damage, including acute replication slowing and phosphorylation of the DNA damage marker histone H2AX. Unusually, the two kinases seemed to cooperate in the checkpoint response, with the loss of either one largely ablating the response, regardless of the type of DNA damage. Thus, T. gondii clearly retains a DNA-damage-responsive checkpoint, but some of its key features differ from the well-studied checkpoint in human cells. AUTHOR SUMMARYToxoplasma is one of the most widespread parasites of humans, with a large proportion of the global population being infected. Infection usually causes no symptoms, but can have severe consequences in pregnant women and immunocompromised people. The Toxoplasma parasite is a single-celled organism, related to other parasites that cause important human diseases like malaria. All these parasites are distinctive in having unusual modes of replication: unlike human cells, they do not replicate by simply splitting in two. Furthermore, drugs to treat toxoplasmosis usually act to suppress the parasites replication. Therefore, it is important to understand how this unusual replication is controlled, both for our fundamental understanding of cell division modes, and to ensure effective drug treatment of toxoplasmosis. Here, we characterised for the first time the proteins that control the Toxoplasma cell cycle by responding to DNA damage. We report that a damage-responsive checkpoint exists, but has some unusual features.

microbiology↗

bioRxiv2024

Histone lactylation: a new epigenetic mark in the malaria parasite Plasmodium

Epigenetic processes play important roles in the biology of the malaria parasite Plasmodium falciparum. Here, we characterised a new epigenetic mark, histone lactylation, recently discovered in humans: it was found in two human malaria parasites, P. falciparum and P. knowlesi, and also in vivo in two rodent malaria models. Histones were lactylated rapidly in response to elevated lactate levels, and rapidly delactylated when lactate levels fell. Thus, this mark is well-placed to act as a metabolic sensor, since severe falciparum malaria characteristically leads to hyperlactataemia. Mass spectrometry showed that lysines on several parasite histones could be lactylated, as well as many non-histone chromatin proteins. Histone lactylation was less abundant and less inducible in P. knowlesi than P. falciparum, suggesting that P. falciparum may have evolved particular epigenetic responses to this characteristic feature of its pathology. Finally, in the rodent model P. yoelii, hyperlactataemia correlated with parasite transcriptomic programmes that suggested metabolic dormancy.

microbiology↗

bioRxiv2023

Effects of the G-quadruplex-binding drugs Quarfloxin and CX-5461 on the malaria parasite Plasmodium falciparum

Plasmodium falciparum is the deadliest causative agent of human malaria. This parasite has historically developed resistance to many drugs, including the current frontline treatments, so new therapeutic targets are needed. Our previous work on guanine quadruplexes (G4s) in the parasites DNA and RNA has highlighted their influence on parasite biology, and revealed G4 stabilising compounds as promising candidates for drug repositioning. In particular, quarfloxin, a former anticancer agent, kills blood-stage parasites at all developmental stages, with fast rates of kill and nanomolar potency. Here we explored the molecular mechanism of quarfloxin and its related derivative CX-5461. In vitro, both compounds bound to P. falciparum-encoded G4 sequences. In cellulo, quarfloxin was more potent than CX-5461, and could prevent establishment of blood-stage malaria in vivo in a murine model. CX-5461 showed clear DNA damaging activity, as reported in human cells, while quarfloxin caused weaker signatures of DNA damage. Both compounds caused transcriptional dysregulation in the parasite, but the affected genes were largely different, again suggesting different modes of action. Therefore, CX-5461 may act primarily as a DNA damaging agent in both Plasmodium parasites and mammalian cells, whereas the complete antimalarial mode of action of quarfloxin may be parasite-specific and remains somewhat elusive.

microbiology↗

bioRxiv2022

Histone lactylation: A new epigenetic axis for host-parasite signalling in malaria?

Epigenetic marks such as histone acetylation and methylation play important roles in the biology and virulence of malaria parasites. Here I report that histone lactylation is also found in these parasites, and speculate on its potential functions. Lactylation is a new epigenetic modification, discovered only in 2019 in human cells. This nascent field has since focussed largely on human biology, but lactyl epigenetic marks could be particularly important in malaria parasites, which are exposed to high and fluctuating lactate levels in their host environment. This is because parasites in the bloodstream respire by glycolysis, producing lactate, and hyperlactataemia is characteristic of severe malarial disease. Therefore, blood lactate could be a signal for the status of the infected host, which could be directly translated to virulence responses via histone lactylation and modulation of parasite gene expression. Responses could include the rate of conversion into sexual transmission stages, the expression of cytoadherence genes - which enhance immune evasion by the parasite but can exacerbate pathology in the host - and the modulation of parasite stress-resistance. Lactylation may soon join acetylation and methylation as a key tool in the epigenetic arsenal of Plasmodium.

microbiology↗

bioRxiv2022

Replication origin mapping in the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum replicates via schizogony: a fundamentally unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. It also has one of the most A/T-biased genomes ever sequenced. Here, we present the first comprehensive study of the specification and activation of DNA replication origins during Plasmodium schizogony. Potential replication origins were found to be abundant, with ORC1-binding sites detected every [~]800 bp throughout the genome. They had no motif enrichment, but were biased towards areas of higher G/C content. Origin activation was then measured at single-molecule resolution via new DNAscent technology that measures fork movement by detecting base analogues BrdU and EdU in DNA sequenced on the Oxford nanopore platform (https://github.com/MBoemo/DNAscent). DNAscent-called origins were found to be much less dense than ORC1-binding sites, with origins activated preferentially in areas of low transcriptional activity. Consistently, replication forks moved fastest through the most lowly transcribed genes, suggesting that conflicts between transcription and origin firing inhibit efficient replication, and that P. falciparum has evolved its S-phase to minimise such conflicts.

microbiology↗

bioRxiv2021

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

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This is a syncytial mode of replication involving asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite replication 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 replications, 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 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 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.

microbiology↗

bioRxiv2021

Dynamics of DNA replication during male gametogenesis in the malaria parasite Plasmodium falciparum

Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle, during which they cycle between mosquito and vertebrate hosts. Sexual reproduction occurs only at the point when parasites move into the mosquito host. It involves specialised, sexually committed cells called gametocytes, which develop very rapidly into mature gametes and then mate inside the mosquito midgut. The gamete development process is unique, involving unprecedentedly fast replication and cell division to produce male gametes. A single male gametocyte replicates its [~]23Mb genome three times over to produce 8 genomes, segregates these into newly-assembled flagellated gamete cells and releases them to seek out female gametes, all within [~]15 minutes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels, yielding several novel observations. Firstly, we confirm that no DNA replication occurs before gametogenesis is triggered, although the origin recognition complex protein Orc1 is abundant even in immature gametocytes. Secondly, between repeated rounds of DNA replication there is no detectable karyokinesis - in contrast to the repeated replicative rounds that occur in asexual schizonts. Thirdly, cytokinesis is clearly uncoupled from DNA replication, and can occur even if replication fails, implying a lack of cell cycle checkpoints. Finally the single-molecule dynamics of DNA replication are entirely different from those in asexual schizonts.

microbiology↗

bioRxiv2021

Plasmodium falciparum GBP2 is a telomere-associated protein that binds to G-quadruplex DNA and RNA

In the early-diverging protozoan parasite Plasmodium, few telomere-binding proteins have been identified and several are unique. Plasmodium telomeres, like those of most eukaryotes, contain guanine-rich repeats that can form G-quadruplex structures. In model systems, quadruplex-binding drugs can disrupt telomere maintenance and some quadruplex-binding drugs are potent anti-plasmodial agents. Therefore, telomere-interacting and quadruplex-interacting proteins may offer new targets for anti-malarial therapy. Here, we report that P. falciparum GBP2 is such a protein. It was identified via Proteomics of Isolated Chromatin fragments, applied here for the first time in Plasmodium. In vitro, PfGBP2 binds specifically to G-rich telomere repeats in quadruplex form and it can also bind to G-rich RNA. In vivo, PfGBP2 partially colocalises with the known telomeric protein HP1 but is also found in the cytoplasm, probably due to its affinity for RNA. Consistently, its interactome includes numerous RNA-associated proteins. PfGBP2 is evidently a multifunctional DNA/RNA-binding factor in Plasmodium.

microbiology↗

bioRxiv2021

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/T-rich transcriptome

Plasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Thus, in this study we produced the in vivo RNA structurome of a protozoan parasite with a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structures of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/T-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This unusual protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.

microbiology↗

bioRxiv2021

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified the formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

microbiology↗