Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this proces

Retrieving sequences of enzymes experimentally characterized but erroneously annotated : the case of the putrescine carbamoyltransferase

BACKGROUND: Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to ressucitate such meaningful data. RESULTS: We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NH(2)-terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences. CONCLUSIONS: We explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

New Insight into the Transcarbamylase Family: The Structure of Putrescine Transcarbamylase, a Key Catalyst for Fermentative Utilization of Agmatine

Transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (CP) to an amine. Although aspartate transcarbamylase and ornithine transcarbamylase (OTC) are well characterized, little was known about putrescine transcarbamylase (PTC), the enzyme that generates CP for ATP production in the fermentative catabolism of agmatine. We demonstrate that PTC (from Enterococcus faecalis), in addition to using putrescine, can utilize L-ornithine as a poor substrate. Crystal structures at 2.5 Å and 2.0 Å resolutions of PTC bound to its respective bisubstrate analog inhibitors for putrescine and ornithine use, N-(phosphonoacetyl)-putrescine and δ-N-(phosphonoacetyl)-L-ornithine, shed light on PTC preference for putrescine. Except for a highly prominent C-terminal helix that projects away and embraces an adjacent subunit, PTC closely resembles OTCs, suggesting recent divergence of the two enzymes. Since differences between the respective 230 and SMG loops of PTC and OTC appeared to account for the differential preference of these enzymes for putrescine and ornithine, we engineered the 230-loop of PTC to make it to resemble the SMG loop of OTCs, increasing the activity with ornithine and greatly decreasing the activity with putrescine. We also examined the role of the C-terminal helix that appears a constant and exclusive PTC trait. The enzyme lacking this helix remained active but the PTC trimer stability appeared decreased, since some of the enzyme eluted as monomers from a gel filtration column. In addition, truncated PTC tended to aggregate to hexamers, as shown both chromatographically and by X-ray crystallography. Therefore, the extra C-terminal helix plays a dual role: it stabilizes the PTC trimer and, by shielding helix 1 of an adjacent subunit, it prevents the supratrimeric oligomerizations of obscure significance observed with some OTCs. Guided by the structural data we identify signature traits that permit easy and unambiguous annotation of PTC sequences

Positional dynamics and glycosomal recruitment of developmental regulators during trypanosome differentiation

African trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies, and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse fly forms is signaled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodeling of their composition and function during the differentiation step, but how this is regulated is not understood. Here we identify a cytological site where the signaling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. In combination, the study provides the first insight into the spatial coordination of signaling pathway components in trypanosomes as they undergo cell-type differentiation.Glycosomes are peroxisome-related organelles that compartmentalize the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite’s needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, Trypanosoma brucei PTP1 (TbPTP1), which dephosphorylates and inhibits a serine threonine phosphatase, TbPIP39, which promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream “stumpy” forms to procyclic forms. Detailed microscopy and live-cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, which defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 min of the initiation of differentiation, TbPIP39 relocates to glycosomes, whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a “stumpy regulatory nexus” (STuRN) that coordinates the molecular components of life cycle signaling and glycosomal development during transmission of Trypanosoma brucei

La putrescine carbamoyltransférase de Streptococcus faecalis. Son intégration dans l'étude de l'évolution des carbamoyltransférases

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Adaptations métaboliques de Trypanosoma brucei en réponse à des variations des conditions intra- et extracellulaires

Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes.Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères

Metabolic adaptations of Trypanosoma brucei in response to changing intra- and extracellular conditions

Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères.Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes