Plasmodium NEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P. berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation

Protein phosphatase 1 regulates atypical mitotic and meiotic division in Plasmodium sexual stages

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time livecell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission

Damage-Induced Mutation Clustering in Gram-Positive Bacteria: Preliminary Data

The phenomenon of a nonrandom distribution of mutations in a genome has been observed for many years. In fact, recent findings have indicated the presence of mutation clusters in different biological systems, including chemically treated yeast, transgenic mice, and human cancer cells. Until now, an asymmetrical distribution of mutations was only described in a single bacterial species. Here, we used ethyl methanesulfonate mutagenesis and a whole-genome sequencing approach to determine if this phenomenon is universal and not confined to Gram-negative bacteria. The Gram-positive bacterium Bacillus subtilis was selected for ethyl methanesulfonate treatment, followed by the next-generation sequencing of several mutagenized B. subtilis genomes. A nonrandom distribution of mutations was observed. This pilot study with a limited number of sequenced clones may indicate not only the universality of the phenomenon of mutation clusters but also the effectiveness of the use of a whole-genome sequencing approach in studying this phenomenon

A genetic barcode of SARS-CoV-2 for monitoring global distribution of different clades during the COVID-19 pandemic

Objective: The SARS-CoV-2 pathogen has established endemicity in humans. This necessitates the development of rapid genetic surveillance methodologies to serve as an adjunct to existing comprehensive, albeit though slower, genome sequencing-driven approaches. Methods: A total of 21,789 complete genomes were downloaded from GISAID on May 28, 2020, for analyses. We have defined the major clades and subclades of circulating SARS-CoV-2 genomes. A rapid sequencing-based genotyping protocol was developed and tested on SARS-CoV-2-positive RNA samples by next-generation sequencing. Results: We describe eleven major mutation events that defined five major clades (G(614), S-84, V-251, I-378, and D-392) of globally-circulating viral populations. The clades can be specifically identified using an 11-nucleotide genetic barcode. An analysis of amino acid variation in SARS-CoV-2 proteins provided evidence of substitution events in the viral proteins involved in both host entry and genome replication. Conclusion: Globally-circulating SARS-CoV-2 genomes could be classified into five major clades based on mutational profiles defined by an 11-nucleotide barcode. We have successfully developed a multiplexed sequencing-based, rapid genotyping protocol for high-throughput classification of major clade types of SARS-CoV-2 in clinical samples. This barcoding strategy will be required to monitor genetic diversity decrease as treatment and vaccine approaches become widely available. (C) 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases

DNA-binding protein PfAP2-P regulates parasite pathogenesis during malaria parasite blood stages.

Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium-specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle