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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Amitochondriates (Life without mitochondrion)

Teaser

Summary

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 â€žmitochondrion-freeâ€œ 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.

Work performed

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 SufB and SufDSU, using the recombinant bacterium E. coli. This allowed us to prove activity of one of them (SufC) in vitro using standard biochemical assay. Once we will obtain the missing two proteins, we plan to reconstitute in vitro the complete pathway. Secondly, we have prepared three mutants of E. coli in which the SufDSU, SufB and SufC genes from M. exilis replaced the native genes of the pathway. In the case of SufB, we have shown that M. exilis protein partially takes over the function of the native E. coli protein, in the other two cases they did not. 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. Finally, we have probed interactions of several pairs of SUF and CIA proteins using the bacterial two hybrid system, but none of them was proved. In summary, we have provided first indirect evidence for the functionality of SufB and SufC protein in M. exilis.

During the work on the second objective, we have proven the existence of mitochondrion in two potential amitochondriate protists â€“ Pelomyxa shiedti and Retortamonas dobelli. The evidence is based on the presence of genes or transcripts for mitochondrial hallmark proteins in the genomes and transcriptomes of these organisms. We are working on characterisation of these organelles further. Secondly, we have performed experiments simulating the situation when and organelle appeared in the cytoplasm of 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 some M. exilis proteins were indeed imported into the hydrogenosomes. This finding was, however, not verified by expression of selected candidates in Trichomonas vaginalis cell. We are elaborating further on these experiments. Finally, we have chosen Entamoeba histolytica, a species of parasitic gut amoeba containing rudimental mitochondrion, as a model for mitochondrial knock out experiments. We are the stage of attempting to downregulate the gene Tom40 essential for the maintenance of the organelle. We have not succeeded yet.

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 roughly characterised the species composition of the prokaryotic component using amplicon 16SrRNA metabarcoding. We have detected more than 50 ribotypes of prokaryotes, the majority representing genera Bacteroidetes and Fusobacterium. Secondly, we are preparing set of plasmids (episomal and integrative) for transient and stable gene expression in M. exilis.

Final results

In the following period we will follow the objectives as outlined in the first part of this report and in more detail in the proposal. We do not see any reason to abandon or markedly modify any of them. On the other hand, we have decided to include two additional aims that in our opinion well complement the rest. (i) Characterisation of metabolic interaction between M. exilis and the prokaryotic component of the culture using metagenomic, metatranscriptomic and metabolomic analysis of the culture 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. (ii) Characterisation of the protein composition of the mitochondrion of Paratrimastix pyriformis. This organism represents the closest relative of M. exilis, which contains a mitochondrion. Understanding of the functions, which this organelle provides to the cell, will be important for hypothesising on the loss of the organelle in M. exilis lineage. We will attempt to determine the protein composition using hyper-LOPIT or LOPIT-DC methods.