Biology subjects

Sievert, M.

Publications and source records attributed to Sievert, M..

3 recordsLinked to original sources

bioRxiv2026

Reduced expression of an essential blood-stage Plasmodium phosphatidylserine synthase does not modulate parasite resistance to PfATP4 inhibitors

Plasmodium falciparum ATP4 mutations A211V and G223R allow parasites to survive the lethal effects of antimalarials PA21A092 (PA92) and cipargamin (CIP), respectively. An A211V mutant line (Dd2A211V) treated with PA92 showed enhanced levels of lipid production, which prompted the idea that components of the phospholipid biosynthesis pathway could be involved in the survival mechanism of PfATP4 mutant parasites. As phosphatidylserine synthase (PfPSS) is the only enzyme that produces phosopholipid phosphatidylserine (PS) in P. falciparum parasites, we hypothesized that PfPSS is both essential for parasite survival and that reduced PfPSS expression would cause resistant PfATP4 mutant parasites to become susceptible to PA92 or CIP. We created a CIP-resistant G223R mutant line (Dd2G223R) via CRISPR-Cas9 and integrated a conditional PfPSS knockdown construct into a Dd2A211V ({downarrow}PSS-Dd2A211V) and our Dd2G223R line ({downarrow}PSS-Dd2G223R). We treated these knockdown lines with PA92 or CIP to determine the half-maximal effective concentration (EC50) of each antimalarial with normal or reduced PfPSS levels. While we found that PfPSS is essential for parasite survival, we did not find any significant alterations to the EC50 values of PA92 or CIP based on the reduced levels of PfPSS in our mutant lines. Although PfPSS does not appear to be involved, other components of the phospholipid production pathway could still affect the resistance mechanism of PfATP4 mutations. Identification of novel targets to counteract the mechanism by which PfATP4 mutant parasites resist lethal drug effects is crucial for the successful application of antimalarials in endemic countries where resistance is on the rise.

microbiology↗

bioRxiv2020

Starvation induces shrinkage of the bacterial cytoplasm

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. colis inner membrane from the cell wall. Shrinkage was accompanied by a [~]17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon-source quality. Robust recovery from starvation required the Tol-Pal system, highlighting the importance of envelope coupling during recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic. Significance statementBacterial cells constantly face nutrient fluctuations in their natural environments. While previous studies have identified gene expression changes upon nutrient depletion, it is much less well known how cellular morphology and cytoplasmic properties respond to shifts in nutrient availability. Here, we discovered that switching fast-growing Escherichia coli cells to nutrient-free conditions results in substantial shrinkage of the inner membrane away from the cell wall, especially at the new pole. Shrinkage was primarily driven by loss of cytoplasmic water contents. Shrinkage was also exhibited by cells naturally entering stationary phase, highlighting its biological relevance across starvation conditions. The membrane-spanning Tol-Pal system was critical for robust entry into and recovery from shrinkage, indicating the importance of cell-envelope homeostasis in surviving nutrient starvation.

microbiology↗

bioRxiv2020

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome

While human induced pluripotent stem cells (iPSCs) offer fascinating prospects for research and clinics, evaluating their genomic stability before applications is of utmost importance. During reprogramming, clonal hiPSC lines derived from the same parental cell population were observed to harbor different mitochondrial DNA (mtDNA) variants and variant heteroplasmy levels. It is unknown to date to which extent this unequal segregation of heteroplasmies between cells arises from mosaicism in the parental cell population, selection on mutated mtDNA molecules on the cellular or organelle level, genetic drift during reduction of mtDNA during reprogramming (genetic bottleneck), or de novo mutations. We analyzed mtDNA variants in 26 clonal iPSC lines by mtDNA sequencing. We did not observe a strong bottleneck or any selection for cells with specific mtDNA variants (clonal eliteness) during reprogramming. iPSC culture expansion, however, selects against putatively actionable mutations in the mitochondrial genome that may affect mitochondrial function and cell metabolism. In contrast, heteroplasmy levels of neutral variants remained stable or increased within clonal iPSC lines during culture expansion. Interestingly, the mtDNA copy number per cell got transiently reduced during targeted differentiation of iPSCs into cardiomyocytes, but mtDNA heteroplasmy levels were not affected. Altogether, our results point towards a scenario in which intra-cellular selection on mtDNA during culture expansion, but not during reprogramming or differentiation, pivotally shapes the mutational landscape of the mitochondrial genome in iPSCs. Other mechanisms such as inter-cellular selection, genetic bottleneck, and genetic drift exert minor impact.

genomics↗