BACKGROUND: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis, a member of the Excavata supergroup of eukaryotes. The mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron-sulfur clusters. RESULTS: In live experiments, no fusion events were observed between the mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in mitosome dynamics. Unlike in mitochondria, division of the mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER-mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. CONCLUSIONS: This study provides the first report on the dynamics of mitosomes. We show that together with the loss of metabolic complexity of mitochondria, mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.

• MeSH
• Biological Evolution MeSH
• Dynamins metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Giardia lamblia cytology   metabolism   MeSH
• Interphase MeSH
• Coenzyme A Ligases metabolism   MeSH
• Long-Chain-Fatty-Acid-CoA Ligase MeSH
• Mitochondrial Dynamics * MeSH
• Mitochondria metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dynamins MeSH
• Coenzyme A Ligases MeSH
• Long-Chain-Fatty-Acid-CoA Ligase MeSH

Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

• MeSH
• Energy Metabolism MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Organelles metabolism   ultrastructure   MeSH
• Oxidation-Reduction MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Gene Expression Regulation MeSH
• Cluster Analysis MeSH
• Sulfur metabolism   MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH
• Protozoan Proteins MeSH
• Sulfur MeSH
• Iron MeSH

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

• MeSH
• Amoeba genetics   metabolism   MeSH
• Cytosol metabolism   MeSH
• Gene Duplication * MeSH
• Entamoeba histolytica metabolism   MeSH
• Nitrogen Fixation genetics   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Sorting Signals MeSH
• Iron-Sulfur Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Sorting Signals MeSH
• Iron-Sulfur Proteins MeSH

Gene duplication is an important evolutionary mechanism and no eukaryote has more duplicated gene families than the parasitic protist Trichomonas vaginalis. Iron is an essential nutrient for Trichomonas and plays a pivotal role in the establishment of infection, proliferation, and virulence. To gain insight into the role of iron in T. vaginalis gene expression and genome evolution, we screened iron-regulated genes using an oligonucleotide microarray for T. vaginalis and by comparative EST (expressed sequence tag) sequencing of cDNA libraries derived from trichomonads cultivated under iron-rich (+Fe) and iron-restricted (-Fe) conditions. Among 19,000 ESTs from both libraries, we identified 336 iron-regulated genes, of which 165 were upregulated under +Fe conditions and 171 under -Fe conditions. The microarray analysis revealed that 195 of 4,950 unique genes were differentially expressed. Of these, 117 genes were upregulated under +Fe conditions and 78 were upregulated under -Fe conditions. The results of both methods were congruent concerning the regulatory trends and the representation of gene categories. Under +Fe conditions, the expression of proteins involved in carbohydrate metabolism, particularly in the energy metabolism of hydrogenosomes, and in methionine catabolism was increased. The iron-sulfur cluster assembly machinery and certain cysteine proteases are of particular importance among the proteins upregulated under -Fe conditions. A unique feature of the T. vaginalis genome is the retention during evolution of multiple paralogous copies for a majority of all genes. Although the origins and reasons for this gene expansion remain unclear, the retention of multiple gene copies could provide an opportunity to evolve differential expression during growth in variable environmental conditions. For genes whose expression was affected by iron, we found that iron influenced the expression of only some of the paralogous copies, whereas the expression of the other paralogs was iron independent. This finding indicates a very stringent regulation of the differentially expressed paralogous genes in response to changes in the availability of exogenous nutrients and provides insight into the evolutionary rationale underlying massive paralog retention in the Trichomonas genome.

• MeSH
• Cysteine Proteases genetics   metabolism   MeSH
• Gene Duplication MeSH
• Expressed Sequence Tags MeSH
• Genome, Protozoan MeSH
• Gene Dosage MeSH
• Gene Library MeSH
• Glycolysis genetics   MeSH
• Evolution, Molecular MeSH
• Iron-Sulfur Proteins genetics   metabolism   MeSH
• Genes, Protozoan * MeSH
• Gene Expression Regulation * MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Transcriptome * MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cysteine Proteases MeSH
• Iron-Sulfur Proteins MeSH
• Iron MeSH

Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group.

• MeSH
• Biological Transport physiology   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Chromatography, Gel MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Organelles metabolism   MeSH
• Porins metabolism   MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Trichomonas vaginalis metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Porins MeSH
• Protozoan Proteins MeSH

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.

• MeSH
• Frataxin MeSH
• Transcription, Genetic MeSH
• Conserved Sequence MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Organelles metabolism   MeSH
• Iron-Binding Proteins genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Iron-Binding Proteins MeSH

Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria ("amitochondriate" protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.

• MeSH
• Ferredoxins genetics   metabolism   MeSH
• Transcription, Genetic MeSH
• Mitochondria MeSH
• Molecular Sequence Data MeSH
• Organelles physiology   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Trichomonas vaginalis genetics   ultrastructure   MeSH
• Hydrogen metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Ferredoxins MeSH
• Protozoan Proteins MeSH
• Hydrogen MeSH

The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 microg/ml [63 microM]). The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads. PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain. The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 microg/ml). Metronidazole-susceptible T. foetus, which expresses very little PDC, is only slightly affected. Omeprazole has the same inhibitory effect on T. foetus cells grown under iron-limited conditions. Similarly to metronidazole-resistant cells, T. foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation.

• MeSH
• Antitrichomonal Agents pharmacology   MeSH
• Iron Deficiencies * MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Kinetics MeSH
• Drug Resistance MeSH
• Metronidazole pharmacology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Omeprazole pharmacology   MeSH
• Pyruvate Decarboxylase drug effects   MeSH
• Tritrichomonas foetus drug effects   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antitrichomonal Agents MeSH
• Enzyme Inhibitors MeSH
• Metronidazole MeSH
• Omeprazole MeSH
• Pyruvate Decarboxylase MeSH