Life without mitochondrion

Project Information

Amitochondriates

Grant agreement ID: 771592

DOI

10.3030/771592

Project closed

EC signature date 18 April 2018

Start date 1 May 2018

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 935 500,00

EU contribution

€ 1 935 500,00

Coordinated by

UNIVERZITA KARLOVA
Czechia

Periodic Reporting for period 4 - Amitochondriates (Life without mitochondrion)

Reporting period: 2022-11-01 to 2023-12-31

Summary of the context and overall objectives of the project

The project investigated the biology of the only eukaryotic organism (Monocercomonoides exilis, Fig. 1) known to lack mitochondrion, an organelle that evolved from bacterial symbiont during the formation of the eukaryotic cell. The research provided the opportunity to show how the cell compensates for the absence of this otherwise essential part. The „mitochondrion-free“ model was useful to study evolutionary processes leading to the emergence and losses of these organelles. It was a basic science project with a low impact on society besides extending our knowledge of cell biology and organellar evolution.

OBJECTIVE 1: To decipher metabolic specialities of M. exilis with a particular focus on the synthesis of FeS clusters. FeS clusters are inorganic functional groups, the synthesis of which is partially performed in mitochondria.
Conclusion: FeS-cluster synthesis in M. exilis is performed by the combination of cytosolic SUF and CIA pathways. All SUF proteins assemble into uniquely large SufBCDSU complex of cca 800 kDa (Fig. 2) which performs all activities of the SUF pathway. Its interaction with CIA pathway is mediated by proteins Nbp35 and Cia1.

OBJECTIVE 2: To tackle 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 emergence or loss of an organelle and (iii) to attempt in vitro preparation of amitochondriate mutants of a selected organism.
Conclusion: Other investigated oxymonads are also amitochondriate indicating that the mitochondrion was lost more than 100 MYA (Fig. 3). The closest relative of oxymonads, Paratrimastix pyriformis, possesses a mitochondrion, whose essential function is the synthesis of one-carbon units. Other investigated eukaryotic lineages (Archamoebae and retortamonads) possess mitochondria, which were characterised (Fig. 4). A small fraction of proteins from the cytosol of amitochondriate M. exilis is imported into the hydrogenosome of T. vaginalis if they are incubated together (Fig. 5).

OBJECTIVE 3: To develop tools that would facilitate studies on M. exilis and transform it into a model species.
Conclusion: We failed to prepare an axenic culture and tools for genetic manipulation. To understand the metabolic connections and dependencies of M. exilis and the prokaryotes in the polyxenic culture, the culture community consisting of >30 prokaryotes was thoroughly characterized (Fig. 6). No clear metabolic link between M. exilis and the bacteria was revealed.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

OBJECTIVE 1
Question 1: How does Monocercomonoides assemble FeS clusters?
We have established that oxymonads and Paratrimastix use for the synthesis of FeS clusters the SUF pathway consisting of SufB, SufC and the fusion protein SufDSU, and the CIA pathway consisting of Nbp35, Nar1, Cia1 Cia 2A and Cia2B proteins, all cytosolic. SUF pathway proteins show all important motifs and residues, perform most important activities, bind necessary cofactors and rescue the growth and/or FeS cluster synthesis in Escherichia coli. All three Suf proteins form in vitro a SufDSUBC complex which probably represents the functional unit in vivo (Fig. 2). This complex probably interacts with Nbp35 and Cia1 proteins of the CIA pathway.

Question 2: What are the major biochemical pathways in the amitochondriate cell?
We planned to solve this question by metabolomics analysis of the axenic culture. As we failed to prepare it, we focused on the processes in the microbial community in which M. exilis grows. This could not provide many details about the metabolism of the amitochondriate cell itself, but we learned that it expresses a wide array of lysozymes and glycosyl hydrolases useful for grazing on bacteria. On the other hand, we uncovered the structure and major metabolic processes in the whole community. It consists of at least 30 species of bacteria (Fig. 6), depending on the external source of organic carbon, which is oxidated and fermented leading to the production of hydrogen which is oxidised to water. Nitrogen compounds are recycled via nitrite ammonification and iron reduction dominates over oxidation.

OBJECTIVE 2
Question 3: Are there other amitochondriate lineages besides oxymonads?
Two other investigated oxymonads are amitochondriate. The mitochondrion-related organelle of the closest relative to oxymonads (Paratrimastix pyriformis) is essential only for the production of one-carbon units. We have investigated the presence of mitochondria in seven other protist species (Retortamonas and Archamoebae) living in low-oxygen environments and detected and characterised mitochondria in all of them (Fig. 4). We have characterised the structure and metabolism of two symbiotic consortia – amoeba Pelomyxa schiedtii with three prokaryotic endosymbionts, oxymonad Streblomastix strix with a dozen of prokaryotic ectosymbionts (Fig. 7).

Question 4: Can we induce the loss of mitosome in the laboratory?
Despite considerable effort, we have failed to prepare a mitochondrial knockout of E. histolytica.

Question 5: What happens to mitochondrion-targeted proteins when the mitochondrion disappears?
Because we failed in Objective 3 and Question 4, we had no experimental system to study what happens to mitochondrial proteins soon after the organelle is lost. To compensate at least partially, we chose a bioinformatics strategy to reveal the long-term fate of mitochondrial proteins in amitochondriate oxymonads. We conclude that no extant oxymonad protein was re-targeted from the mitochondrion into another compartment, and they were all lost after the organelle disappeared.

Question 6: How does the cell proteome react to the emergence of mitochondrial compartment?
We showed that at least 14 proteins from M. exilis cytosol are imported into hydrogenosomes of Trichomonas vaginalis if co-incubated (Fig. 4). We speculate that this mimics the situation when the import into the early mitochondrion was evolving and supports the hypothesis that the mistargeting of a small fraction of proteins from the host cell contributed to the emerging mitochondria.

OBJECTIVE 3: OXYMONADS AS A MODEL AMITOCHONDRIATE CELL LINE
We failed to axenise the culture and we did not succeed with the genetic engineering of the cells.

DISSEMINATION OF THE RESULTS
The results were disseminated via conference contributions, theses and publications. The team members defended nine theses and 22x participated in conferences where they presented the results of the project. Finally, the results were published in 13 open-access publications in impacted journals and two preprints currently under review.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Besides the progress in knowledge summarised above we pushed some methods beyond the state-of-the-art, namely the introduction of the bulk import system into hydrogenosomes (Fig. 4), the application of LOPIT proteomics to non-axenic protist culture and we also refined methods of (single-cell) metagenomics and transcriptomics for characterising the culture community and symbiotic consortia.

Figure 5: Import of M. exilis proteins in T. vaginalis hydrogenosomes

Figure 3: Proposed scenario of the loss of mitochondrion in oxymonads

Figure 2: Suf pathway of M. exilis

Figure 1: Monocercomonoides exilis

Figure 4: Overview of the Pelomyxa schiedti MRO metabolism

Figure 7: Oxymonad Streblomastix strix and its ectosymbiotic bacteria

Figure 6: Prokaryotic composition of the culture community