Genome-scale reconstruction of the Streptococcus pyogenes M49 metabolic network reveals growth requirements and indicates potential drug targets.

Genome-scale metabolic models comprise stoichiometric relations between metabolites, as well as associations between genes and metabolic reactions and facilitate the analysis of metabolism. We computationally reconstructed the metabolic network of the lactic acid bacterium Streptococcus pyogenes M49. Initially, we based the reconstruction on genome annotations and already existing and curated metabolic networks of Bacillus subtilis, Escherichia coli, Lactobacillus plantarum and Lactococcus lactis. This initial draft was manually curated with the final reconstruction accounting for 480 genes associated with 576 reactions and 558 metabolites. In order to constrain the model further, we performed growth experiments of wild type and arcA deletion strains of S. pyogenes M49 in a chemically defined medium and calculated nutrient uptake and production fluxes. We additionally performed amino acid auxotrophy experiments to test the consistency of the model. The established genome-scale model can be used to understand the growth requirements of the human pathogen S. pyogenes and define optimal and suboptimal conditions, but also to describe differences and similarities between S. pyogenes and related lactic acid bacteria such as L. lactis in order to find strategies to reduce the growth of the pathogen and propose drug targets.

Using a genome-scale metabolic model of Enterococcus faecalis V583 to assess amino acid uptake and its impact on central metabolism.

Increasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially l-glutamine and l-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.

Acetate formation in the energy metabolism of parasitic helminths and protists.

Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation.

Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms.

Procyclic forms of Trypanosoma brucei isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates, do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically preadapted to adequate functioning in the tsetse fly.

Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica.

Fasciola hepatica contains anaerobically functioning mitochondria that produce acetate and propionate, the main endproducts excreted by this parasite. The final reactions in the pathways leading to these endproducts are performed by acetate:succinate CoA-transferase (ASCT) and propionate:succinate CoA-transferase (PSCT), respectively. The enzymes catalysing these essential reactions in anaerobic mitochondria are still not characterized, nor are the corresponding genes identified. Here we describe the identification of the gene that codes for the F. hepatica ASCT. The F. hepatica gene was heterologously expressed and studies on the corresponding enzyme activity showed that the enzyme is indeed a transferase and uses a ping-pong bi-bi reaction mechanism, like most other known CoA-transferases. This F. hepatica CoA-transferase was shown to be a true transferase and not a hydrolase, as it needs an acceptor for optimal activity. Our studies demonstrated that the F. hepatica ASCT can use other CoA-acceptors than succinate, such as propionate, acetate and butyrate, and is in fact a short-chain acyl-CoA-transferase. We further showed that this F. hepatica CoA-transferase can also catalyze the PSCT reaction, which is responsible for the production of propionate. Analysis of the amino acid sequence of F. hepatica clearly indicated the presence of a mitochondrial targeting sequence, and in CHO cells the enzyme is indeed present in the mitochondrial fraction. F. hepatica ASCT is the first ASCT identified in anaerobic mitochondria. It is homologous to the hydrogenosomal ASCT we earlier identified in Trichomonas vaginalis, but not to the ASCT present in the aerobic mitochondria of Trypanosoma brucei.

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization.

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

Energy metabolism of bloodstream form Trypanosoma theileri.

Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions.

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.