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.

• Keywords
• CTR copper transporters, DJ-1, Naegleria fowleri, Naegleria gruberi, alternative NADH dehydrogenase, alternative oxidase, copper, electron transport chain,
• Publication type
• Journal Article MeSH

Naegleria fowleri is a single-cell organism living in warm freshwater that can become a deadly human pathogen known as a brain-eating amoeba. The condition caused by N. fowleri, primary amoebic meningoencephalitis, is usually a fatal infection of the brain with rapid and severe onset. Iron is a common element on earth and a crucial cofactor for all living organisms. However, its bioavailable form can be scarce in certain niches, where it becomes a factor that limits growth. To obtain iron, many pathogens use different machineries to exploit an iron-withholding strategy that has evolved in mammals and is important to host-parasite interactions. The present study demonstrates the importance of iron in the biology of N. fowleri and explores the plausibility of exploiting iron as a potential target for therapeutic intervention. We used different biochemical and analytical methods to explore the effect of decreased iron availability on the cellular processes of the amoeba. We show that, under iron starvation, nonessential, iron-dependent, mostly cytosolic pathways in N. fowleri are downregulated, while the metal is utilized in the mitochondria to maintain vital respiratory processes. Surprisingly, N. fowleri fails to respond to acute shortages of iron by inducing the reductive iron uptake system that seems to be the main iron-obtaining strategy of the parasite. Our findings suggest that iron restriction may be used to slow the progression of infection, which may make the difference between life and death for patients.

• MeSH
• Cell Respiration MeSH
• Adaptation, Physiological * MeSH
• Mitochondria metabolism   MeSH
• Naegleria fowleri genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Trace Elements metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Trace Elements MeSH
• Iron MeSH

Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.

• MeSH
• Cytosol metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Iron-Sulfur Proteins chemistry   metabolism   MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Trypanosoma brucei brucei growth & development   metabolism   MeSH
• Trypanosomiasis metabolism   parasitology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Protozoan Proteins MeSH