A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite''s glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)_≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.     

24-Methylenecyclopropane steroidal inhibitors: A Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei

A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15 ± 5 μM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24β-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (k_cat/k_inact = 0.13 min^− 1/0.08 min^− 1; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC–MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases.     

Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3′-5′ exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.     

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei’s survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC₅₀) of PCF was 1.0 ± 0.2 μM for Isoxyl and 5 ± 2 μM for 10-TS, whereas BSF appeared more susceptible with EC₅₀ values 0.10 ± 0.03 μM (Isoxyl) and 1.0 ± 0.6 μM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents’ survival.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

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.African trypanosomatids comprise various pleomorphic trypanosome species that proliferate in the bloodstream of their mammalian hosts as long slender bloodstream form (BSF) trypanosomes, and at the peak of parasitemia they differentiate into nondividing short stumpy form trypanosomes (1). After being ingested during a bloodmeal by a tsetse fly (Glossina sp.), short stumpy form trypanosomes differentiate into procyclic form (PCF) trypanosomes, which actively multiply and colonize the midgut of the fly. Subsequently, PCF Trypanosoma brucei migrates to the salivary glands while undergoing a complex differentiation (22). Here, attached epimastigote forms start multiplying, after which nondividing metacyclic trypomastigotes develop. The life cycle of T. brucei is completed when these metacyclic trypomastigotes are injected into a mammal through the bite of an infected fly, after which they transform into long slender BSF trypanosomes. During this life cycle, trypanosomes encounter different environments to which they have adapted, resulting in distinct stages, characterized by morphological as well as metabolic changes. Long slender BSF trypanosomes degrade glucose by glycolysis and excrete pyruvate as the sole metabolic end product (12, 13, 23). On the other hand, PCF trypanosomes do not excrete pyruvate but degrade glucose to acetate and succinate as main end products (25). Krebs cycle activity was thought previously to be present in trypanosomatids, at least in insect stages of some African trypanosomatids (3, 9, 10, 12, 21). However, this presumed flux through the Krebs cycle is supported only poorly by direct experimental evidence and was based mainly on the presence of certain enzyme activities. Although genes for all enzymes of this cycle are indeed present in the genome and expressed in the insect stages, recent studies revealed that at least in T. brucei, the cycle is not used for the complete oxidation of acetyl-coenzyme A (CoA) to carbon dioxide (2, 26). Instead, parts of the cycle are most likely used in anabolic pathways, such as gluconeogenesis and fatty acid formation, and also for the final steps in the degradation of amino acids (26). It is possible that the reported discrepancies on the presence or absence of full-circle Krebs cycle activity are caused by differences in the number of passages through mice after the isolation of the strain from the field. Such passages may have been ongoing for many years, during which the parasites were continuously propagated as BSF trypanosomes. Furthermore, most insect form trypanosomes that were investigated up to now have been propagated for many years as PCF trypanosomes in rich culture media. Hence, the reported discrepancies could be due to differences between freshly differentiated PCF trypanosomes and those well adapted to in vitro culture, and the absence of an active Krebs cycle in PCF trypanosomes could be the result of an adaptation caused by the prolonged in vitro culturing. To investigate these possibilities, we analyzed the glucose metabolism of PCF T. brucei directly after isolation from the midguts of tsetse flies. We also studied freshly differentiated PCF trypanosomes from the AntAR 1 strain, a T. brucei strain that has had a minor history of animal passaging since its field isolation (15, 17).To investigate the cause of the conflicting reports on Krebs cycle activity in PCF trypanosomes, we first analyzed the effect of environmental factors by comparing the carbohydrate metabolism of PCF trypanosomes well adapted to in vitro culturing and PCF trypanosomes isolated from their natural environment, the midguts of tsetse flies. These experiments were performed with PCF TREU 927 T. brucei, a pleomorphic strain that has been thoroughly characterized and is still able to infect Glossina morsitans, performing a complete physiological life cycle (2). For the infection of tsetse flies, male G. morsitans flies originating from the colony maintained at the Institute of Tropical Medicine in Antwerp, Belgium, were infected with procyclic TREU 927 T. brucei by in vitro membrane feeding and subsequently maintained for 10 days by feeding on rabbit blood (15). Then, flies were dissected on a sterile glass slide and the infected midguts were isolated and incubated for at least 30 min at 28°C in SDM-79 medium that was gently rotated. After sedimentation of the midguts by gravity, insect gut debris was removed by centrifugation at 300 × g for 5 min. PCF trypanosomes were then isolated from the collected supernatant by centrifugation at 1,500 × g for 10 min. Since PCF trypanosomes could not be isolated from the midgut without minor amounts of contaminating insect gut material, such as gut cells and debris, we also investigated the glucose metabolism of this fraction. Analysis of metabolic end products produced from [6-¹⁴C]glucose in this control incubation of insect gut debris, which also contained minor amounts of trypanosome cells, showed the formation of ¹⁴C-labeled pyruvate, CO₂, acetate, and lactate (Fig. ​(Fig.1A).1A). Minor amounts of lactate were also produced in the incubations with PCF trypanosomes isolated from the midgut, which also contained minor amounts of insect gut debris. Since lactate is not an excreted end product of T. brucei, this labeled lactate is indicative for the glucose degradation activity of insect gut debris. Therefore, end product formation in the incubations with PCF trypanosomes isolated from the midgut was corrected for end products produced by the contaminating insect gut debris by subtracting all produced lactate and the calculated accompanying amounts of other end products produced in the insect gut debris incubation. The metabolic incubations with PCF trypanosomes directly after isolation from the tsetse midgut showed that these trypanosomes degrade glucose to the same metabolic end products, acetate, succinate, and pyruvate, as the in vitro culture-adapted PCF trypanosomes (Fig. ​(Fig.1A).1A). Furthermore, the ratio of acetate and succinate produced by PCF trypanosomes isolated from the midgut were similar to that of in vitro-cultured PCF trypanosomes (Fig. ​(Fig.1A).1A). On the other hand, a major difference was observed in the amount of glucose consumed since the PCF trypanosomes isolated from the midguts of tsetse flies consumed 16-fold less glucose than PCF trypanosomes that were derived from in vitro cultures. This difference in glucose consumption can probably be explained by our observation that both motility and especially growth of PCF trypanosomes isolated from the midgut were significantly reduced compared to the in vitro culture-derived PCF trypanosomes. Apparently, the environmental conditions in the midgut of the fly did affect the PCF trypanosomes, but they did not significantly alter the metabolic pathways used for energy metabolism. However, PCF trypanosomes isolated from the midgut of the fly excreted more pyruvate (Fig. ​(Fig.1A),1A), which suggests that pyruvate is a more important metabolic end product for PCF trypanosomes under physiological conditions than acknowledged thus far. Most importantly, however, just like continuously in vitro-cultured ones, PCF trypanosomes isolated from the midgut of the fly did not degrade [6-¹⁴C]glucose to labeled CO₂ (Fig. ​(Fig.1A),1A), which demonstrates the absence of a functional Krebs cycle in these tsetse fly-derived PCF trypanosomes.Open in a separate windowFIG. 1.Radioactive end products of [6-¹⁴C]glucose metabolism of procyclic TREU 927 T. brucei cells grown in vitro or isolated from the midguts of tsetse flies (A) and that of AntAR 1 T. brucei during differentiation of BSF to PCF trypanosomes (B). (A) The results of a single experiment for PCF trypanosomes isolated from the midgut and for insect gut debris and the mean + the standard deviation (SD) of three parallel incubations for in vitro-cultured PCF trypanosomes are shown. Total end product formation from [6-¹⁴C]glucose was 2.08 ± 0.19 μmol/h per 10⁸ cells and 0.23 μmol/h per 10⁸ cells for in vitro-cultured and midgut-isolated PCF trypanosomes, respectively, and was calculated using the number of trypanosome cells present at the beginning of the incubation. End product formation in the incubation with PCF trypanosomes isolated from the midgut was corrected for end products produced by contaminating insect gut debris (see text for details). (B) Metabolic incubations using 6-¹⁴C-labeled glucose were performed during differentiation from short stumpy BSF trypanosomes to insect stage PCF trypanosomes. Incubations with PCF trypanosomes were started at 24, 48, and 96 h after induction of differentiation (PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4, respectively); means + SDs of three parallel incubations are shown (for the short stumpy form, six incubations in two independent experiments). Total glucose consumption in incubations with long slender BSF trypanosomes, short stumpy BSF trypanosomes, PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4 was 4.8, 3.4, 1.5, 1.1, and 0.79 μmol/h per 10⁸ cells, respectively. Excreted labeled end products shown in panels A and B were analyzed as described previously (25) and are expressed as the percentage of the total amount of radioactive end products produced (in the incubation of gut debris, one other unidentified end product was produced, which explains why this total in the figure does not add up to 100%). The decrease in pyruvate production between long slender and short stumpy BSF trypanosomes as well as the increase in acetate production is significant as calculated using an unpaired t test (P < 0.01 for pyruvate and P < 0.001 for acetate).Although TREU 927 T. brucei is a pleomorphic trypanosome strain, it cannot be excluded that these trypanosomes have adapted their energy metabolism during the substantial period that this strain has been cultured in vitro. Therefore, we also studied the carbohydrate metabolism of freshly transformed PCF of the T. brucei AntAR 1 strain, a well-characterized pleomorphic strain that is close to the wild isolate (17). To investigate the energy metabolism of these freshly differentiated PCF trypanosomes, AntAR 1 BSF trypanosomes were harvested from the blood of infected immune-suppressed NMRI mice as described previously (16) and either directly incubated with [6-¹⁴C]glucose or differentiated to PCF trypanosomes, by addition of 6 mM cis-aconitate and incubation at 27°C (7). These trypanosomes were then incubated with [6-¹⁴C]glucose at different time points after the initiation of differentiation. Our experiments (Fig. ​(Fig.1B)1B) confirmed that differentiation of trypanosomes from BSF to PCF is accompanied by a metabolic shift in excreted end products from pyruvate to acetate and succinate (3, 14, 25). This metabolic shift during differentiation of BSF to PCF trypanosomes was complete after 1 to 2 days (Fig. ​(Fig.1B),1B), which is in agreement with previous observations (9). A subsequent switch in medium from HMI-9, a medium used to culture BSF T. brucei, to SDM-79, a medium used for the culture of PCF T. brucei, did not result in further changes in excreted end products (data not shown).Our experiments, however, did not show any significant production of labeled CO₂ and certainly not the massive increase in CO₂ formation upon differentiation of BSF into PCF trypanosomes that was reported in a comparable study by Durieux et al. (9). We cannot exclude that this difference in Krebs cycle activity between our study and that of Durieux et al. is caused by a strain difference, but since the AntAR 1 strain we used can be considered to be close to the field isolate, the results presented here are indicative of wild-type T. brucei metabolism and strongly suggest that a functional Krebs cycle is absent in PCF T. brucei cells in vivo.Next to the absence of carbon dioxide formation via Krebs cycle activity during differentiation of BSF to PCF trypanosomes, our metabolic experiments also demonstrated that acetate accounted for 30% of the glucose-derived excreted labeled end products in freshly isolated BSF AntAR 1 T. brucei cells (Fig. ​(Fig.1B).1B). This is a surprising observation since BSF trypanosomes are reported to rely on glycolysis only and to excrete pyruvate and minor amounts of glycerol (12, 13, 23). However, the BSF trypanosomes that we tested in our incubations were predominantly short stumpy BSF cells, whereas nearly all previously performed metabolic studies of BSF trypanosomes were performed with long slender BSF cells. In order to investigate whether differentiation from long slender to short stumpy form trypanosomes indeed shifts the metabolism toward acetate formation, we analyzed the energy metabolism of BSF trypanosomes harvested from mice at two different time points after infection. At day 4 after infection, predominantly long slender BSF trypanosomes were isolated (94% long slender versus 6% short stumpy), whereas at day 7 after infection, predominantly short stumpy BSF trypanosomes were isolated (92% short stumpy versus 8% long slender). Analysis of glucose-derived metabolic end products from incubations with BSF AntAR 1 trypanosomes isolated at day 4 or at day 7 after infection showed that short stumpy BSF trypanosomes indeed produce significant amounts of acetate as an end product of glucose metabolism (Fig. ​(Fig.1B).1B). In the incubations with predominantly long slender BSF AntAR 1 T. brucei cells, some acetate was also produced, but this relatively small amount of acetate formation can be explained by the presence of a certain amount of short stumpy cells. Although the incubations were started with nearly 95% long slender BSF cells, BSF cells from the AntAR 1 strain are highly pleomorphic and rapidly differentiate to short stumpy forms during in vitro culture conditions. Therefore, increasing amounts of short stumpy form T. brucei were formed during our incubations (up to 40 to 50% at the end of incubation), which accounts for the amount of acetate formed during these incubations.Since acetate production in Trypanosomatidae is catalyzed by the mitochondrial enzyme acetate:succinate CoA transferase (ASCT), which was previously shown not to be expressed in in vitro-cultured BSF T. brucei (20), we examined the ASCT enzyme activity in lysates derived from either over 92% short stumpy cells or 94% long slender cells. These experiments showed that the ASCT enzyme is present in short stumpy BSF trypanosomes in an amount equivalent to around 15% of that of PCF trypanosomes (Fig. ​(Fig.2).2). This is in agreement with the observation that acetate is a more prominent excreted end product in PCF trypanosomes than in short stumpy BSF cells. On the other hand, ASCT activity was nearly absent in long slender BSF trypanosomes (Fig. ​(Fig.2),2), which confirms the conclusion that in our incubations acetate is not produced by long slender BSF trypanosomes but by short stumpy BSF trypanosomes.Open in a separate windowFIG. 2.ASCT activity in total lysates of T. brucei AntAR 1. Enzymatic activity of ASCT was determined in total lysates derived from cultures containing predominantly long slender BSF trypanosomes (BSF LS; 94%), predominantly short stumpy BSF trypanosomes (BSF SS; 92%), or exclusively PCF trypanosomes (PCF). Shown are the means + standard deviations of three experiments.Hence, our experiments show that short stumpy BSF trypanosomes do not only degrade glucose by glycolysis but additionally produce acetate. Acetate formation in trypanosomes occurs via the mitochondrial enzyme ASCT and involves transfer of a CoA moiety from acetyl-CoA to succinate, yielding succinyl-CoA (24). This succinyl-CoA can then be converted back into succinate by succinyl-CoA synthetase, a reaction concomitantly converting ADP in ATP (6, 24). Therefore, our observations that short stumpy BSF trypanosomes produce acetate and express ASCT demonstrate that these stages in addition to glycolysis also use a mitochondrial pathway for the degradation of glucose and production of ATP.Multiple mitochondrial adaptations have been reported to occur during the transition from long slender BSF to short stumpy BSF T. brucei. Differential gene expression and the formation of cristea in the inner mitochondrial membrane have been shown to occur during this transition (8, 11, 19). Furthermore, the trypanosomal homologue of complex I of the respiratory chain is expressed in short stumpy BSF trypanosomes (4, 5, 18). Our experiments show that this more elaborate composition of the electron transport chain is also used by this stage, as the production of acetate implies that acetyl-CoA is formed, which is catalyzed by the pyruvate dehydrogenase complex and results in the production of NADH inside the mitochondrion. This means that either complex I or the alternative NADH dehydrogenase is active in this stage (18). Moreover, our experiments show that the previously reported mitochondrial adaptations in short stumpy BSF trypanosomes are not restricted to morphological changes and to changes in the composition of the electron transport chain but also result in a functionally altered energy metabolism.In conclusion, the data described in this paper demonstrate the absence of a functional Krebs cycle in the mitochondria of PCF T. brucei, isolated from the tsetse midgut or freshly differentiated from BSF trypanosomes. Furthermore, we show that short stumpy BSF T. brucei cells produce large amounts of acetate. Therefore, the mitochondria of short stumpy trypanosomes are metabolically divergent from the mitochondria in long slender BSF T. brucei cells. These results are consistent with prior work (4, 5, 8, 11). The functional changes might be a preadaptation that allows short stumpy BSF T. brucei to function in the intestines of infected tsetse flies and enables them to differentiate further into PCF trypanosomes.     

Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1^−/− parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein TbHEN1 methylates siRNA 3′ ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.     

Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs

Background

Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the trans-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against Leishmania amazonensis.

Methodology/Principal Findings

Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-L. amazonensis activity with IC₅₀ values of 18 μM, 65 μM, 95 μM and 65 μM for promastigotes, respectively. For intracellular amastigotes, the IC₅₀ values of the analogs were 33.2 μM, 45 μM, 29 μM and 30.5 μM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death.

Conclusion/Significance

Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg²⁺. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn''t affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.     

Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (K _i = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC₅₀ = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available.     

TbTRF suppresses the TERRA level and regulates the cell cycle-dependent TERRA foci number with a TERRA binding activity in its C-terminal Myb domain

Telomere repeat-containing RNA (TERRA) has been identified in multiple organisms including Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. VSG is expressed exclusively from subtelomeric expression sites, and we have shown that telomere proteins play important roles in the regulation of VSG silencing and switching. In this study, we identify several unique features of TERRA and telomere biology in T. brucei. First, the number of TERRA foci is cell cycle-regulated and influenced by TbTRF, the duplex telomere DNA binding factor in T. brucei. Second, TERRA is transcribed by RNA polymerase I mainly from a single telomere downstream of the active VSG. Third, TbTRF binds TERRA through its C-terminal Myb domain, which also has the duplex DNA binding activity, in a sequence-specific manner and suppresses the TERRA level without affecting its half-life. Finally, levels of the telomeric R-loop and telomere DNA damage were increased upon TbTRF depletion. Overexpression of an ectopic allele of RNase H1 that resolves the R-loop structure in TbTRF RNAi cells can partially suppress these phenotypes, revealing an underlying mechanism of how TbTRF helps maintain telomere integrity.     

Dexamethasone and Azathioprine Promote Cytoskeletal Changes and Affect Mesenchymal Stem Cell Migratory Behavior

Glucocorticoids and immunosuppressive drugs are commonly used to treat inflammatory disorders, such as inflammatory bowel disease (IBD), and despite a few improvements, the remission of IBD is still difficult to maintain. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have emerged as regulators of the immune response, and their viability and activation of their migratory properties are essential for successful cell therapy. However, little is known about the effects of immunosuppressant drugs used in IBD treatment on MSC behavior. The aim of this study was to evaluate MSC viability, nuclear morphometry, cell polarity, F-actin and focal adhesion kinase (FAK) distribution, and cell migratory properties in the presence of the immunosuppressive drugs azathioprine (AZA) and dexamethasone (DEX). After an initial characterization, MSCs were treated with DEX (10 μM) or AZA (1 μM) for 24 hrs or 7 days. Neither drug had an effect on cell viability or nuclear morphometry. However, AZA treatment induced a more elongated cell shape, while DEX was associated with a more rounded cell shape (P < 0.05) with a higher presence of ventral actin stress fibers (P < 0.05) and a decrease in protrusion stability. After 7 days of treatment, AZA improved the cell spatial trajectory (ST) and increased the migration speed (24.35%, P < 0.05, n = 4), while DEX impaired ST and migration speed after 24 hrs and 7 days of treatment (-28.69% and -25.37%, respectively; P < 0.05, n = 4). In conclusion, our data suggest that these immunosuppressive drugs each affect MSC morphology and migratory capacity differently, possibly impacting the success of cell therapy.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.     

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening

Trypanothione synthetase (TryS) catalyses the synthesis of N¹,N⁸-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC₅₀ from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC₅₀ values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC₅₀ from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC₅₀ against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS’s synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.     

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2′-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.     

Genetic and chemical evaluation of Trypanosoma brucei oleate desaturase as a candidate drug target

Background

Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites.

Methodology

Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied.

Principal Findings

Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC₅₀ values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC₅₀ values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential.

Conclusions/Significance

As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.     

Curiouser and Curiouser: The Macrocyclic Lactone,Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms

Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced R_max, no change in EC₅₀). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.