Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited

Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes

The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis

Correction: How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell

In the Abstract, the seventh sentence is incorrect. The correct sentence reads: Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while other ATP-dependent processes account for the remaining ~37% of the ATP consumption, with translation being the most expensive process. In the penultimate sentence in the final paragraph of the Introduction, the value “62%” should be “63%.” In the Results section, the fourth paragraph should be preceded by the subtitle “The cost of genome duplication.” The first sentence of the sub-section “Energy cost of sugar nucleotides used in the synthesis of the VSG coat” is incorrect. The correct sentence reads: In the BSF of T. brucei, the major surface protein is the VSG, which is highly glycosylated and linked to the membrane by GPI anchors. The seventh sentence of the sub-section “ATP requirement for transmembrane transport” is incorrect. The correct sentence reads: It is worth mentioning that Stouthamer did not consider the costs of taking up glucose, which could be relevant for many prokaryotes but not for BSF T. brucei where glucose transport happens by facilitated diffusion [115,116]. The penultimate sentence in the sub-section “How much ATP hydrolysis is required to maintain the mitochondrial inner membrane potential (ΔCm)?” is incorrect. The correct sentence reads: We hypothesize that this mitochondrial substrate-level phosphorylation system is the main source of intramitochondrial ATP, and it can provide sufficient ATP to maintain the ΔCm [22,157], despite its relatively low capacity for producing ATP [10]. In Table 11, the BSF Trypanosoma brucei “Total” value should be 6.00 x 1011. The seventeenth sentence of the third Discussion paragraph is incorrect. The correct sentence reads: As we considered 2 ATP molecules being spent per base polymerized, an average transcript length of 2,800 nt, and an average RNA synthesis of 1.2 RNAs/h (estimated in [51]) the estimated ATP expenditure for nuclear transcription is ~2.9 x 107 ATP molecules (0.5% of the total ATP expenditure for the completion of a cell cycle) (Fig 1, S5 Table). The fourth sentence of the fourth Discussion paragraph is incorrect. The correct sentence reads: Therefore, taken together, translation and protein turnover demand 59.6% of the ATP budget (Table 13). The penultimate sentence of the fourth Discussion paragraph is incorrect. The correct sentence reads: These calculations do not include the cost of the synthesis of sugar nucleotides used for the glycosylation of surface proteins (mostly VSGs) and anchoring. The ninth sentence of the seventh Discussion paragraph is incorrect. The correct sentence reads: To estimate the total percentage of the budget used for flagellar beating, we considered the highest value obtained, which resulted in the consumption of 9.5% of ATP produced (Table 13).</p

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle