Final Report Summary - BHMLOEVOFUNC (Evolution and function of the Blastocytis mitochondrion-like organelle)
Blastocystis is a unicellular human intestinal parasite, of controversial pathogenicity. Since Blastocystis is a strict anaerobe, it was thought to possess hydrogenosomes, anaerobic adenosine triphosphate (ATP)-generating organelles related to mitochondria. In contrast to typical hydrogenosomes, blastocystis has mitochondrion-related organelle (MRO)s with cristae, a transmembrane potential and deoxyribonucleic acid (DNA). Previous work has demonstrated a mitochondrial genome and bioinformatics analyses of expressed sequence tag (EST)s have identified several putative mitochondrial and hydrogenosomal proteins. These data suggest that Blastocystis MROs may be some kind of a metabolic intermediate between mitochondria and hydrogenosomes.
To test the predicted functions of these MROs, my research objectives were to characterise the mitochondrion related organelle of Blastocystis using a combination of molecular and cell biological tools along with bioinformatics tools. Some of my main aims included:
1. generation of a more comprehensive Blastocystis EST data set by pyrosequencing methods and search these data to fill in the missing links on the metabolic maps of this organelle;
2. analyse Blastocystis' organellar proteome to understand how it has adapted to an anaerobic lifestyle and compare it with other eukaryotes harbouring mitochondria-like organelles;
3. test the hypothesis that Blastocystis has retained functional Fe-S cluster biosynthesis pathways; and
4. identify how environmental conditions affect the metabolism of Blastocystis MROs.
Since the beginning of the project, I have established several projects for the investigation of some of the functions (metabolic pathways) of Blastocystis MROs, including:
1. New 454 sequencing of a new EST library
In collaboration with Dr Eleni Gentekaki (at the Andrew Roger's laboratory - outgoing phase), we have extracted total ribonucleic acid (RNA) from Blastocystis cells (strain NandII; subtype 1), grown under anaerobic and aerobic conditions, in order to generate a more comprehensive EST data set and fill in the missing links on the metabolic maps of this organelle. The extracted RNA was sent to Vertis (Germany) for the preparation of a cDNA library. The library was subsequently sent to Génome Québec Innovation Centre (Canada) for a titanium sequencing. The sequencing results provided us with 122 405 raw reads which resulted into 15 781 clusters with the use of the Newbler assembler. In an attempt to get a more thorough coverage along with longer clusters, we have assembled the previous EST results along with the new data. Using the MIRA program we assembled 137 157 raw reads into 12 019 clusters, varying in length from 49 bp to 3627 bp. Preliminary analyses of these new EST clusters provided us with new in silico predictions of the functions of Blastocystis MRO, along with potential adaptations of the parasite to an anaerobic lifestyle. Even though these are preliminary analyses, some of the proteins that were predicted are: additional members of mitochondrial carrier family (MCF) proteins, several enzymes responsible for pyruvate metabolism (two pyruvate:ferredoxin oxidoreductase (PFO) proteins, a pyruvate:NADP+ oxidoreductase (PNO) enzyme, all subunits of pyruvate dehydrogenase (PDH)), one of the maturases of (FeFe)-hydrogenase, five homologues of the iron-sulphur proteins of complex II (succinate dehydrogenase 2 (SDH2)), components of several of its predicted pathways including iron-sulphur (Fe-S) cluster biosynthesis, amino acid metabolism and mitochondrial import. In summary, and in comparison with the first broad transcriptomic and genomic studies of Blastocystis that have identified 115 and 360 genes respectively, encoding putative mitochondrial and hydrogenosomal proteins, we have identified at least 412 putative MRO proteins. The localisation of these proteins would potentially be confirmed with the proteomic studies (see below).
2. Proteomics of purified Blastocystis MRO
One of the strategies that I have used to investigate the functions of Blastocystis MRO involves purifying the organelle for localisation, biochemical and proteomic analyses. Since previous established organellar protocols have not been successful for the purification of the Blastocystis MRO (certain strain: NandII), I have decided to follow an alternative strategy. Blastocystis organelles were purified using an optimised OptiPrep gradient protocol. The purity of the organellar fraction was assessed using customed made antibodies against mitochondrial proteins of Blastocystis (PFO, T-protein, Hydrogenase) and a cytosolic protein (SufCB; see below). Purified organelles were subsequently treated with trypsin and the resulting peptides were separated by high performance liquid chromatography on a nano high-performance liquid chromatography (HPLC) Ultimate 3000 instrument with sample preconcentration flow manager. Around 150 proteins were identified using the automated spotter Probot; with subsequent use of tandem matrix-assisted laser desorption ionisation time-of-flight analyser (matrix-assisted laser desorption / ionisation (MALDI) time-of-flight (TOF) / TOF); AB/MDS Sciex 4800 TOF / TOF analyser). The available softwares (Mascot and Paragon) were used for data analyses. Preliminarily data confirm the presence of the predicted biosynthetic pathways from the ESTs analyses (see above), but further investigation should take place in the near future.
3. The Blastocystis Fe-S cluster machinery
In the clustered Blastocystis ESTs, I have identified homologues of the core proteins for the mitochondrial iron sulfur cluster (ISC) machinery (e.g. IscU, IscS, Frataxin, Ferredoxin, Isa2, mrs3/4, mitochondrial Hsp70 and glutaredoxin), which are also found in the majority of other MROs. In the case of the Isa2 (a scaffold protein with uncerain functions), I have demonstrated that Blastocystis Isa2 is localised in the mitochondria of Trypanosoma brucei and, by employing Trypanosoma brucei RNAi knockdowns, I have demonstrated that the Blastocystis Isa2 homologue can functionally replace the Trypanosoma Isa1/2 knock-downs, suggesting overlapping functions of these proteins in diverse eukaryotes.
In addition, we discovered a new (sulphur mobilisation machinery; SufCB) Fe-S cluster assembly machinery in Blastocystis that has been acquired by lateral gene transfer from methanoarchaea, which I have decided to investigate its localisation and function along with the further investigations on the mitochondrial counterparts. For this reason, I have initiated a collaboration with Prof. Julius Lukes' laboratory (University of South Bohemia, Czech Republic) in order to characterise the mitochondrial Fe-S cluster assembly using the in vitro system with Trypanosoma brucei knock-downs. As part of this collaboration I have visited the aforementioned laboratory, where I have learned the techniques and transfer them back to the Roger lab (where I have characterised two of the proteins discussed in the paper). I have also initiated a collaboration with the laboratories of Frederic Barras and Mark Fontecave (France) for the characterisation of the SufCB. In parallel, I have localised several of the components of the Fe-S cluster machineries (SufCB in the cytosol and ISC in the MROs) in Blastocystis and I was involved in the design of all the experiments that took place.
In summary, results of the above analyses (including more results coming in the near future) shed light on the unknown function of the mitochondrion in this anaerobic parasitic organism, thereby elucidating the evolutionary history and diversity of both anaerobic metabolism and mitochondria or related organelles within microbial eukaryotes.
1. Stechmann, A. et al., Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes. Curr Biol, 2008. 18(8): p. 580-5.
2. Denoeud, F. et al., Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biol, 2011. 12(3): p. R29.
3. Nasirudeen, A. M. and K. S. Tan, Isolation and characterisation of the mitochondrion-like organelle from Blastocystis hominis. J Microbiol Methods, 2004. 58(1): p. 101-9.
4. Tsaousis, A. D. et al., Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis. Proc Natl Acad Sci U S A, 2012. 109(26): p. 10426-31.
5. Long, S. et al., Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues. Mol Microbiol, 2011. 81(6): p. 1403-18.
To test the predicted functions of these MROs, my research objectives were to characterise the mitochondrion related organelle of Blastocystis using a combination of molecular and cell biological tools along with bioinformatics tools. Some of my main aims included:
1. generation of a more comprehensive Blastocystis EST data set by pyrosequencing methods and search these data to fill in the missing links on the metabolic maps of this organelle;
2. analyse Blastocystis' organellar proteome to understand how it has adapted to an anaerobic lifestyle and compare it with other eukaryotes harbouring mitochondria-like organelles;
3. test the hypothesis that Blastocystis has retained functional Fe-S cluster biosynthesis pathways; and
4. identify how environmental conditions affect the metabolism of Blastocystis MROs.
Since the beginning of the project, I have established several projects for the investigation of some of the functions (metabolic pathways) of Blastocystis MROs, including:
1. New 454 sequencing of a new EST library
In collaboration with Dr Eleni Gentekaki (at the Andrew Roger's laboratory - outgoing phase), we have extracted total ribonucleic acid (RNA) from Blastocystis cells (strain NandII; subtype 1), grown under anaerobic and aerobic conditions, in order to generate a more comprehensive EST data set and fill in the missing links on the metabolic maps of this organelle. The extracted RNA was sent to Vertis (Germany) for the preparation of a cDNA library. The library was subsequently sent to Génome Québec Innovation Centre (Canada) for a titanium sequencing. The sequencing results provided us with 122 405 raw reads which resulted into 15 781 clusters with the use of the Newbler assembler. In an attempt to get a more thorough coverage along with longer clusters, we have assembled the previous EST results along with the new data. Using the MIRA program we assembled 137 157 raw reads into 12 019 clusters, varying in length from 49 bp to 3627 bp. Preliminary analyses of these new EST clusters provided us with new in silico predictions of the functions of Blastocystis MRO, along with potential adaptations of the parasite to an anaerobic lifestyle. Even though these are preliminary analyses, some of the proteins that were predicted are: additional members of mitochondrial carrier family (MCF) proteins, several enzymes responsible for pyruvate metabolism (two pyruvate:ferredoxin oxidoreductase (PFO) proteins, a pyruvate:NADP+ oxidoreductase (PNO) enzyme, all subunits of pyruvate dehydrogenase (PDH)), one of the maturases of (FeFe)-hydrogenase, five homologues of the iron-sulphur proteins of complex II (succinate dehydrogenase 2 (SDH2)), components of several of its predicted pathways including iron-sulphur (Fe-S) cluster biosynthesis, amino acid metabolism and mitochondrial import. In summary, and in comparison with the first broad transcriptomic and genomic studies of Blastocystis that have identified 115 and 360 genes respectively, encoding putative mitochondrial and hydrogenosomal proteins, we have identified at least 412 putative MRO proteins. The localisation of these proteins would potentially be confirmed with the proteomic studies (see below).
2. Proteomics of purified Blastocystis MRO
One of the strategies that I have used to investigate the functions of Blastocystis MRO involves purifying the organelle for localisation, biochemical and proteomic analyses. Since previous established organellar protocols have not been successful for the purification of the Blastocystis MRO (certain strain: NandII), I have decided to follow an alternative strategy. Blastocystis organelles were purified using an optimised OptiPrep gradient protocol. The purity of the organellar fraction was assessed using customed made antibodies against mitochondrial proteins of Blastocystis (PFO, T-protein, Hydrogenase) and a cytosolic protein (SufCB; see below). Purified organelles were subsequently treated with trypsin and the resulting peptides were separated by high performance liquid chromatography on a nano high-performance liquid chromatography (HPLC) Ultimate 3000 instrument with sample preconcentration flow manager. Around 150 proteins were identified using the automated spotter Probot; with subsequent use of tandem matrix-assisted laser desorption ionisation time-of-flight analyser (matrix-assisted laser desorption / ionisation (MALDI) time-of-flight (TOF) / TOF); AB/MDS Sciex 4800 TOF / TOF analyser). The available softwares (Mascot and Paragon) were used for data analyses. Preliminarily data confirm the presence of the predicted biosynthetic pathways from the ESTs analyses (see above), but further investigation should take place in the near future.
3. The Blastocystis Fe-S cluster machinery
In the clustered Blastocystis ESTs, I have identified homologues of the core proteins for the mitochondrial iron sulfur cluster (ISC) machinery (e.g. IscU, IscS, Frataxin, Ferredoxin, Isa2, mrs3/4, mitochondrial Hsp70 and glutaredoxin), which are also found in the majority of other MROs. In the case of the Isa2 (a scaffold protein with uncerain functions), I have demonstrated that Blastocystis Isa2 is localised in the mitochondria of Trypanosoma brucei and, by employing Trypanosoma brucei RNAi knockdowns, I have demonstrated that the Blastocystis Isa2 homologue can functionally replace the Trypanosoma Isa1/2 knock-downs, suggesting overlapping functions of these proteins in diverse eukaryotes.
In addition, we discovered a new (sulphur mobilisation machinery; SufCB) Fe-S cluster assembly machinery in Blastocystis that has been acquired by lateral gene transfer from methanoarchaea, which I have decided to investigate its localisation and function along with the further investigations on the mitochondrial counterparts. For this reason, I have initiated a collaboration with Prof. Julius Lukes' laboratory (University of South Bohemia, Czech Republic) in order to characterise the mitochondrial Fe-S cluster assembly using the in vitro system with Trypanosoma brucei knock-downs. As part of this collaboration I have visited the aforementioned laboratory, where I have learned the techniques and transfer them back to the Roger lab (where I have characterised two of the proteins discussed in the paper). I have also initiated a collaboration with the laboratories of Frederic Barras and Mark Fontecave (France) for the characterisation of the SufCB. In parallel, I have localised several of the components of the Fe-S cluster machineries (SufCB in the cytosol and ISC in the MROs) in Blastocystis and I was involved in the design of all the experiments that took place.
In summary, results of the above analyses (including more results coming in the near future) shed light on the unknown function of the mitochondrion in this anaerobic parasitic organism, thereby elucidating the evolutionary history and diversity of both anaerobic metabolism and mitochondria or related organelles within microbial eukaryotes.
1. Stechmann, A. et al., Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes. Curr Biol, 2008. 18(8): p. 580-5.
2. Denoeud, F. et al., Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biol, 2011. 12(3): p. R29.
3. Nasirudeen, A. M. and K. S. Tan, Isolation and characterisation of the mitochondrion-like organelle from Blastocystis hominis. J Microbiol Methods, 2004. 58(1): p. 101-9.
4. Tsaousis, A. D. et al., Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis. Proc Natl Acad Sci U S A, 2012. 109(26): p. 10426-31.
5. Long, S. et al., Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues. Mol Microbiol, 2011. 81(6): p. 1403-18.