Project description:Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including two basal bodies (equivalent to centrioles), two flagella (equivalent to cilia), and a cytoplasmic microtubule array. This full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of cytoskeletal, metabolism, signaling and stress response genes. Naegleria gruberi (strain NEG) was grown in association with Kleibsiella pneumoniae on solid media. Cells were prepared and differentiated using standard protocols, and harvested at 0, 20, 40, 60 and 80 minutes after initiation of differentiation.
Project description:Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including two basal bodies (equivalent to centrioles), two flagella (equivalent to cilia), and a cytoplasmic microtubule array. This full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of cytoskeletal, metabolism, signaling and stress response genes. Naegleria gruberi (strain NEG) was grown in association with Kleibsiella pneumoniae on solid media. Cells were prepared and differentiated using standard protocols, and harvested at 0, 20, 40, 60 and 80 minutes after initiation of differentiation. Three independent biological replicates were obtained from differentiating N. gruberi. Each replicate series included the following timepoints: 0, 20, 40, 60 and 80 minutes after initiation of differentiation.
Project description:Although copper is an essential nutrient crucial for many biological processes, an excessive concentration can be toxic and lead to cell death. The metabolism of this two-faced metal must be strictly regulated at the cell level. In this study, we investigated copper homeostasis in two related unicellular organisms: nonpathogenic Naegleria gruberi and the “brain-eating amoeba” Naegleria fowleri. We identified and confirmed the function of their specific copper transporters securing the main pathway of copper acquisition. Adjusting to different environments with varying copper levels during the life cycle of these organisms requires various metabolic adaptations. Using comparative proteomic analyses, measuring oxygen consumption, and enzymatic determination of NADH dehydrogenase, we showed that both amoebas respond to copper deprivation by upregulating the components of the branched electron transport chain: the alternative oxidase and alternative NADH dehydrogenase. Interestingly, analysis of iron acquisition indicated that this system is copper-dependent in N. gruberi but not in its pathogenic relative. Importantly, we identified a potential key protein of copper metabolism of N. gruberi, the homolog of human DJ-1 protein, which is known to be linked to Parkinson’s disease. Altogether, our study reveals the mechanisms underlying copper metabolism in the model amoeba N. gruberi and the fatal pathogen N. fowleri and highlights the differences between the two amoebas.
Project description:The formation of mitochondria has enabled eukaryotes to thrive and evolve into multitude of unicellular and multicellular species. The nature of the early mitochondria and its relationship to the ancestral host cell is still unknown. Using a phylogenomic approach we have discovered the presence of core subunits of type II secretion pathway (T2SS) in several unicellular eukaryotes (protists) belonging to jakobids, hetereloboseans and malawimonads. We show that the proteins carry mitochondrial signal sequences and are localized in the protist mitochondria.The mitochondria of Nagleria gruberi were isolated by differential centrifugation and analysed by mass spectrometry. The data analyses revealed about 1000 proteins enriched in the mitochondrial fraction including the components of T2SS.
Project description:Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.