Investigating the cellular localization and functional role of iron-sulphur cluster assembly machinery in Naegleria gruberi

Naegleria gruberi is a eukaryotic microbe that belongs to the group of excavates. It is a free-living eukaryotic unicellular amoeboflagellate, with the capacity to change life forms (amoeba, flagellate, cyst) and shift from aerobic to anaerobic respiration. N. gruberi is an important organism to study for many reasons. Firstly,because of its close relationship with the brain-eating amoeba Naegleria fowleri, which causes Primary Amoebic Meningoencephalitis (PAM). Also, because of its unique internal and external traits. It has the capacity of transitioning to different life forms such as amoeba, flagellate, and cyst, in accordance with its external conditions. Additionally, it holds a crucial position to the phylogenetic tree and possesses similarities to the Last Eukaryotic Common Ancestor (LECA), reflecting its evolution. Amongst organisms there are various cellular and biochemical pathways that have been conserved. Some of them are the Iron- Sulphur (Fe-S) cluster biosynthetic pathways, which are of great importance. The Fe-S cluster mechanisms have been found in all the organisms studied to date and they are crucial for their survival. Eukaryotic cells typically harbour two of these mechanisms. These mechanisms are the Iron-Sulphur cluster assembly mechanism (ISC) and the Cytosolic Iron-Sulphur cluster assembly mechanism (CIA). The ISC is found in both eukaryotic and prokaryotic organisms, and it is responsible for the biosynthesis of Fe-S clusters and their transportation and incorporation of a variety of proteins. It therefore plays an important part in electron transport, enzyme activity, and overall mitochondrial function. On the other hand, the CIA mechanism is found exclusively in eukaryotic organisms, and it involves the maturation of Fe-S clusters, and their incorporation into nuclear and cytosolic proteins. Therefore, it is important for several cellular processes, such as DNA repair. Other Fe-S cluster mechanisms include the Sulphur Mobilization (SUF) mechanism and the Nitrogen Fixation (NIF) mechanism. These mechanisms are usually found in prokaryotes. The SUF mechanism is usually activated under extreme conditions, such as oxidative stress, while the NIF mechanism is responsible for the production of Fe-S cluster for the proteins involved in nitrogen fixation.

The aim of this study was to understand the evolution, location, and characteristics of the ISC mechanism within N. gruberi. For this a combination of bioinformatics and wet-lab experimental approaches were used. The bioinformatic analyses includes the alignment of the N. gruberi proteins to the same proteins of other organisms as well as the construction of phylogenetic trees. Those tests point out the conservation of the ISC proteins amongst different species, displaying that some proteins were more conserved than others, in addition to their place in the phylogenetic tree and an initial view to their evolutionary path. All the above showed that the N. gruberi proteins we targeted are closer to those of prokaryotic organisms. Lastly, bioinformatic analysis gave an initial idea of the localization of the proteins within the cell, which places some of them in the mitochondria and others in the cytosol. Additionally, through bioinformatics, the presence of proteins from the other Fe-S cluster mechanisms within the cell was tested. Proteins of the NIF system seem to be absent from the cell. Some proteins were similar to those of the SUF machinery, but further research is important to confirm or reject this hypothesis. Further experimental techniques including immunolocalization placed the proteins of the ISC machinery in both the mitochondria and the cytosol of the organism, proving that the pathway can be translocated outside the mitochondria if necessary.