How Many Is Enough? - Challenges of Multinucleated Cell Division in Malaria Parasites

Abstract

Regulating the number of progeny generated by replicative cell cycles is critical for any organism to best adapt to its environment. Classically, the decision whether to divide further is made after cell division is completed by cytokinesis and can be triggered by intrinsic or extrinsic factors. Contrarily, cell cycles of some species, such as the malaria-causing parasites, go through multinucleated cell stages. Hence, their number of progeny is determined prior to the completion of cell division. This should fundamentally affect how the process is regulated and raises questions about advantages and challenges of multinucleation in eukaryotes. Throughout their life cycle Plasmodium spp. parasites undergo four phases of extensive proliferation, which differ over three orders of magnitude in the amount of daughter cells that are produced by a single progenitor. Even during the asexual blood stage proliferation parasites can produce very variable numbers of progeny within one replicative cycle. Here, we review the few factors that have been shown to affect those numbers. We further provide a comparative quantification of merozoite numbers in several P. knowlesi and P. falciparum parasite strains, and we discuss the general processes that may regulate progeny number in the context of host-parasite interactions. Finally, we provide a perspective of the critical knowledge gaps hindering our understanding of the molecular mechanisms underlying this exciting and atypical mode of parasite multiplication.

Keywords: Plasmodium; cell division; fungi; malaria; mitosis; multinucleated; polyploidy.

Figure 1

Nuclear and genome copy numbers throughout Plasmodium spp. life cycle stages. (A) Schematic depiction of approximate total number of nuclei produced during the proliferative life cycle stages of P. falciparum as well as the highest observed ploidy (n) or genome copy number within individual nuclei. (B) Number of merozoites produced in P. falciparum and P. knowlesi. To count merozoites, late stage parasites were enriched by magnetic purification or Nycodenz density gradient. Parasites were incubated with the cyclic GMP-dependent protein kinase (PKG) inhibitor ML10 (25 nM in P. falciparum strains; 125 nM in P. knowlesi) for 3 h to prevent egress, increasing the percentage of post-mitotic parasites. After preparation of blood smears parasites were fixed with 4% paraformaldehyde for 20 min at 37°C. Before imaging, cells were stained with Hoechst. Microscopy was performed on a Leica TCS SP8 scanning confocal microscope with Lightning (LNG) module. LNG enables automated adaptive deconvolution after acquisition resulting in super-resolution images. For each Plasmodium strain, 100 cells were imaged. Merozoites were counted in a single-blind mode by three independent researchers using Fiji. The mean merozoite number for each individual cell was taken to create the violin plot. The centered black dot represents the median. High density of nuclei in P. falciparum made it difficult to assess the definitive final number of nuclei. This counting error might contribute to the presence of many uneven numbers in P. falciparum strains, while in P. knowlesi almost exclusively even numbers were counted. For statistical analysis, a Welch-ANOVA test was performed, and individual lines were compared with a post hoc Games-Howell test using R studio. For P. falciparum strains, FCR3 and 3D7 (p = 2.4x10^-4) as well as FCR3 and Dd2 (p = 4.2x10^-2) showed significant differences.

Figure 2

Three models highlighting variables that can contribute to progeny number. Variation in progeny number can be explained by changes in several cellular parameters. Relative number of nuclei are plotted against the total duration of blood stage development, which is divided in growth, division, and budding phase. The longest division time is marked, which starts with the first and ends with the last nuclear division. Time axes are neither to scale nor proportional. Three models provide a visual representation of changing a single determinant which limits the final progeny number and explain the variability within a single parasite population. Extrinsic or intrinsic factors can contribute to either an increase (green) or a decrease (red) of progeny number by (A) influencing the pre-set target number (counting), (B) initiating division at different times (timing) or (C) altering the rate of division of individual parasites.