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Life without mitochondrion

Periodic Reporting for period 3 - Amitochondriates (Life without mitochondrion)

Reporting period: 2021-05-01 to 2022-10-31

The project investigates the biology of the only eukaryotic organism (Monocercomonoides exilis) known to lacks mitochondrion, an organelle that evolved from bacterial symbiont already during the formation of the eukaryotic cell. Discovery of such unicellular flagellate provides oportunity to show how the cell compensates absence of this otherwise essential part by tweeking its internal processes. It also provides us potentially useful „mitochondrion-free“ cellular model to study general cell biology as well as evolutionary processes leading to the emergence and losses of organelles.

It is a basic science project with low impact on society besides extending our knowledge on cell biology and organellar evolution.

The project has three main objectives:

1) To decipher metabolic specialities of Monocercomonoides exilis with particular focus on the synthesis of FeS clusters. FeS clusters are inorganic functional groups in many enzymes, the synthesis of which is partially performed in mitochondria. It has become clear from the genomic study that in M. exilis the synthesis is achieved by a very different way, probably using a combination of two enzymatic pathways (SUF and CIA), which have never been reported to work together.

2) To tackle some questions related to mitochondrial evolution, namely (i) to establish the presence/absence of mitochondrion in other potentially amitochondriate lineages, (ii) to show by direct experiment how the proteins in the cytoplasm react to an emergence or loss of an organelle and (iii) to attempt in vitro preparation of amitochondriate mutants of selected organism.

3) To develop tools that would facilitate studies on Monocercomonoides exilis and would transform it to a model species. By such tools I mean cultivation method without prokaryotic contaminants, methods for influencing of expression of its genes and methods for introducing or deleting of genes.
I will organise the results by objectives.

During the work on the first objective, we have, firstly, produced all proteins of SUF and CIA pathways, with the exceptions of SufDSU, using the recombinant bacterium E. coli. This allowed us to prove the activity of SufC in vitro using standard biochemical assay and to prove the formation of a stable SufBC complex. Using a bacterial two-hybrid system, we have also confirmed the interactions between SufC and SufB but also between SufC and SufDSU. Interestingly, the M. exilis SufC was also able to interact with the E. coli homologue of SufB and SufS and, consistently, we were able to show that M. exilis SufDSU was able to substitute the function of SufS in E. coli knockouts suggesting it has a cysteine desulfurase activity. Similarly, we have prepared yeast mutants, in which the genes from CIA pathway were substituted by respective genes from M. exilis, but none of them was able to substitute the function of the yeast protein. In summary, we have provided the first evidence for the functionality of SUF pathway in M. exilis.

During the work on the second objective, we have proven the existence and characterised the mitochondria in two potential amitochondriate protists – Pelomyxa shiedti and Retortamonas dobelli, both of them resembling the reduced mitochondria in related species. Secondly, we have performed experiments simulating the situation when an organelle appeared in the cytoplasm of the amitochondriate cell by incubation of hydrogenosomes (derivate of mitochondrion) isolated from T. vaginalis in the cytoplasmic extract of M. exilis. Proteomic analyses of the hydrogenosomes after incubation suggested that at least 24 M. exilis proteins were indeed imported into the hydrogenosomes. We are validating these results by expressing the tagged versions of these proteins in T. vaginalis. In 4 out of 9 validated cases, we have confirmed the import but found that the efficiency of the import is low. We are working on the validation of the remaining candidates and performing analogous experiments with the mitochondrion of T. brucei. Finally, we have chosen Entamoeba histolytica, a species of parasitic gut amoeba containing rudimental mitochondrion, as a model for mitochondrial knock out experiments. Have established the protocol for measuring the concentration of PAPS, essential product of E. histolytica mitosomes, and prepared a cell line expressing cytosolic PAPS synthase. Unfortunately, we have failed so far in the knockdown of mitosomal maintenance genes or inhibition of the mitosomes in any other way.

During the work on the third objective, we have so far not succeeded in axenisation (purifying from prokaryotic contamination) of the culture. At least we have characterised the species composition of the prokaryotic component using amplicon 16SrRNA metabarcoding and metagenomic sequencing. We have obtained more than 24 prokaryotic complete or partial genomes, the most represented phyla were Bacteroidetes and Firmicutes. We are continuously attempting to transform M. exilis using various alternatives of plasmids and we are preparing constructs for CRISPR/Cas9 knock out system.
In the following period we will follow the objectives as outlined in the first part of this report and more detail in the proposal. We do not see any reason to abandon or markedly modify any of them. On the other hand, due to our failure to axenise the culture we have decided to modify the strategy for studying metabolomics of the M. exilis. Instead of metabolomic analysis of the pure culture, we have decided to investigate the ecosystem in the tube using metagenomic/metatranspriptomic and metabolomic approach at various stages of growth. This might uncover potentially interesting connections between the eukaryote and prokaryotes in the test tube and might help with further axenisation attempts. On top of the original plan, we have also included characterisation of the protein composition of the mitochondrion of Paratrimastix pyriformis using LOPIT proteomics. This organism represents the closest relative of M. exilis, which contains a mitochondrion. Understanding the functions, which this organelle provides to the cell, will be important for hypothesising on the loss of the organelle in M. exilis lineage. This experiment is already done, and analyses almost finished. We predict 30 proteins into the organelle and hypothesise that the production of formate is its key role.
Longitudinal section of the cell of Monocercomonoides exilis by transmission electron microscope