A rapid approach for in locus overexpression of Trypanosoma brucei proteins.

Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.

Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes.

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.

NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi.

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.

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis.

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas vaginalis. The kinetic properties and thermostability of T. vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T. vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.

Proteomic and transcriptomic analysis of heart failure due to volume overload in a rat aorto-caval fistula model provides support for new potential therapeutic targets - monoamine oxidase A and transglutaminase 2.

BACKGROUND: Chronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF. METHODS: We analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip. RESULTS: In the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs. CONCLUSIONS: The differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.

The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis.

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 ß-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.

The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis.

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation.

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein-tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.

The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica.

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.

Pyridine-N-oxide analogues of DOTA and their gadolinium(III) complexes endowed with a fast water exchange on the square-antiprismatic isomer.

Two macrocyclic ligands derived from H4dota containing three acetate pendant arms and one 2-methylpyridine-N-oxide coordinating unit were synthesized. The ligand H3do3apy(NO) (H3L1, 10-[(1-oxidopyridin-2-yl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) contains an unsubstituted pyridine-N-oxide ring; the ligand H4do3apy(NO-C) (H4L2, 10-[(4-carboxy-1-oxidopyridin-2-yl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) is functionalized with a carboxylic group in the 4 position of the pyridine ring to allow attachment to other molecules. The ligands form octadentate (N4O4 environment) Ln(III) complexes, with one water molecule completing the coordination sphere in a capping position. The complexes are present in solution exclusively as square-antiprismatic isomers over the whole lanthanide series. The introduction of the carboxylic group to the pyridine-N-oxide unit in H4L2 has no significant effect on the hydration number (q = 1) and the water exchange rate of the [Gd(H2O)(L2)]- complex compared to the parent [Gd(H2O)(L1)] complex (water residence times: tauM = 39 ns for [Gd(H2O)(L1)] and tauM = 34 ns for [Gd(H2O)(L2)]- at 298 K).

Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.

Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.

Cattle pathogen tritrichomonas foetus (Riedmüller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species.

A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonasfoetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus-T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commission on Zoological Nomenclature for ruling under its plenary power.