Biology subjects

Johnson, M. K.

Publications and source records attributed to Johnson, M. K..

4 recordsLinked to original sources

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

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↗

bioRxiv2023

LETHAL COVID-19 ASSOCIATES WITH RAAS-INDUCED INFLAMMATION FOR MULTIPLE ORGAN DAMAGE INCLUDING MEDIASTINAL LYMPH NODES

Lethal COVID-19 outcomes are most often attributed to classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes which significantly correlated with COVID-19 viral load, include many canonical innate immune genes. However, 22 much less studied "non-canonical" genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, with a tightly associated mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies validate the transcriptomic findings showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap findings in SARS-CoV-2 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early variants and later SARS-CoV-2 strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.

immunology↗

bioRxiv2017

Challenges for Bayesian Model Selection of Dynamic Causal Models

Achieving a mechanistic explanation of brain function requires understanding causal relationships among regions. A relatively new technique to assess effective connectivity in fMRI data is Dynamic Causal Modeling (DCM). As DCM is more frequently used, it becomes increasingly important to further validate the technique and understand its limitations. With DCM, Bayesian Model Selection (BMS) is used to select the most likely causal model. We conducted simulations to test the degree to which BMS is robust to two types of challenges when applied to DCMs, those inherent to data (Category 1) and those inherent to model space (Category 2). Category 1 challenges tested properties of the data (low signal-to-noise, different response magnitudes and shapes across regions) that could either blur the distinction between models or potentially bias model selection. These challenges are impossible or difficult to measure and control in real data, so investigating their effect upon BMS through simulation is critical. Category 2 challenges tested properties of model space that create subsets of confusable models. Our results suggest that given data that conform to the prior assumptions of DCM, BMS is robust to challenges from Category 1. However, in the face of Category 2 challenges (when a more homogenous model space was tested) the false positive rate rose above an acceptable level. We show that such errors are neither trivial nor easily avoided with existing approaches. However, we argue that it is possible to detect Category 2 challenges, and avoid inappropriate interpretations by conducting simulations prior to applying DCM.\n\nAcronyms

neuroscience↗