Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

African trypanosomes are early divergent protozoan parasites responsible for high mortality and morbidity as well as a great economic burden among the world's poorest populations. Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated immune evasion mechanism. Their nuclear organization and mechanisms for gene expression control present several conventional features but also a number of striking differences to the mammalian counterparts. Some of these unorthodox characteristics, such as lack of controlled transcription initiation or enhancer sequences, render their monogenic antigen transcription, which is critical for successful antigenic variation, even more enigmatic. Recent technological developments have advanced our understanding of nuclear organization and gene expression control in trypanosomes, opening novel research avenues. This review is focused on Trypanosoma brucei nuclear organization and how it impacts gene expression, with an emphasis on antigen expression. It highlights several dedicated sub-nuclear bodies that compartmentalize specific functions, whilst outlining similarities and differences to more complex eukaryotes. Notably, understanding the mechanisms underpinning antigen as well as general gene expression control is of great importance, as it might help designing effective control strategies against these organisms.

Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

The anti-leishmania effects of HIV peptidase inhibitors (PIs) have been widely reported; however, the biochemical target and mode of action are still a matter of controversy in Leishmania parasites. Considering the possibility that HIV-PIs induce lipid accumulation in Leishmania amazonensis, we analysed the effects of lopinavir on the lipid metabolism of L. amazonensis promastigotes. To this end, parasites were treated with lopinavir at different concentrations and analysed by fluorescence microscopy and spectrofluorimetry, using a fluorescent lipophilic marker. Then, the cellular ultrastructure of treated and control parasites was analysed by transmission electron microscopy (TEM), and the lipid composition was investigated by thin-layer chromatography (TLC). Finally, the sterol content was assayed by gas chromatography–mass spectrometry (GC/MS). TEM analysis revealed an increased number of lipid inclusions in lopinavir-treated cells, which was accompanied by an increase in the lipophilic content, in a dose-dependent manner. TLC and GC–MS analysis revealed a marked increase of cholesterol-esters and cholesterol. In conclusion, lopinavir-induced lipid accumulation and affected lipid composition in L. amazonensis in a concentration–response manner. These data contribute to a better understanding of the possible mechanisms of action of this HIV-PI in L. amazonensis promastigotes. The concerted action of lopinavir on this and other cellular processes, such as the direct inhibition of an aspartyl peptidase, may be responsible for the arrested development of the parasite.

Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ∼12 million people worldwide, with ∼2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 μM) inhibited parasite growth in a dose-dependent manner (IC50=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3β-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3β-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0·5 of 0·1 μm and a Hill number (nH)=2·8±0·4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7·0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0·25±0·19 μm and Km2=29·6±6·8 μm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

The mini-exon gene repeats from three different strains of Endotrypanum schaudinni have been amplified by the polymerase chain reaction (PCR). Sequence analysis of the cloned products shows the gene and intergenic region to be identical in two of the strains (LV86 and M6159); the intergenic region from the mini-exon gene of the third strain (LV59) is significantly different. The LV86 gene and an intergenic probe from the LV59 mini-exon gene do not cross-hybridize with the mini-exon gene from New World Leishmania species. These data provide the basis of a PCR assay to detect E. schaudinni and distinguish it from New World Leishmania species, and which should be applicable to epidemiological studies in insect vectors and mammalian reservoirs. The identification of two different mini-exon gene repeats in E. schaudinni isolates is indicative of further strain variation within this species.

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5′-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4·7±1·0 μm but a fraction of about 40–50% was insensitive to CrATP. Remarkably, at low substrate concentration (0·2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0·33±0·10 μm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2′ disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Until recently it had generally been assumed that apoptosis and other forms of programmed cell death evolved during evolution of the metazoans to regulate growth and development in these multicellular organisms. However, recent research is adding strength to the original phenotypic observations described almost a decade ago which indicated that some parasitic protozoa may have evolved a cell death pathway analogous to the process described as apoptosis in metazoa. Here we explore the implications of a programmed cell death pathway in the African tsetse-transmitted trypanosomes.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5·5 to 9·0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

In previous studies, we showed that Herpetomonas samuelpessoai produced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 from Leishmania spp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by living H. samuelpessoai cells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS–PAGE to determine the secretory protein profile and on gelatin-SDS–PAGE to identify the proteolytic activity. The results demonstrated that H. samuelpessoai secreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6·0 at 37 °C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin of L. amazonensis demonstrated the presence of similar molecules on the cell-surface of H. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid of H. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide from H. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from the H. samuelpessoai surface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed in Leishmania.

The spliced leader-associated (SLA1) RNA is a trypanosome-specific small RNA with unknown function. SLA1 carries a Sm-like site, and is associated with core Sm proteins. Here we found that SLA1 belongs to a family of hairpin-containing RNAs that are implicated in directing pseudouridylation. A potential for base-pair interaction between SLA1 and spliced leader (SL) RNA agrees with the canonical rules for guiding pseudouridylation on SL RNA. Direct RNA analysis showed that this uridine is indeed pseudouridylated in the SL RNA of Leptomonas collosoma, Leishmania major, and Trypanosoma brucei. This position is conserved in all trypanosomatid SL RNAs. Mutations introduced in the SL RNA to disrupt the interaction domain of SLA1/SL RNA abolished the formation of the pseudouridine. SLA1 is localized both to the nucleolus and nucleoplasm. This study solves a long-standing question regarding the function of this novel RNA and describes the first H/ACA RNA, which, unlike all other pseudouridine guides, is also a bona fide snRNA.

The three trypanosomatid genome projects have employed common strategies which include: analysis of pulsed-field gel electrophoretic chromosomal karyotypes; physical mapping using big DNA (cosmid, pacmid P1, bacterial artificial chromosome, yeast artificial chromosome) libraries; partial cDNA sequence analysis to develop sets of expressed sequence tags (ESTs) for gene discovery and use as markers in physical mapping; genomic sequencing; dissemination of information through development of web-sites and ACeDB-based fully integrated databases; and establishment of functional genomics programmes to maximize useful application of genome data. Highlights of the projects to date have been the demonstration that, despite extensive chromosomal size polymorphisms for diploid homologues within Africa trypanosomes, T. cruzi or Leishmania, the physical linkage groups for markers on each chromosome are retained across all isolates/species studied within each group. For African trypanosomes, detailed analysis of chromosome 1 has demonstrated that repetitive sequences and the two retroposon-like elements RIME and INGI are localized to a defined region at one end of the chromosome, with the bulk of the central region of the chromosome containing genes coding for expressed proteins. Comparative mapping shows that, although subtelomeric changes account for a large proportion of the polymorphism in chromosome size in African trypanosomes, there are significant expansions and contractions in regions across the entire chromosome. The highlight of the genomic sequencing projects has been the demonstration of just 2 putative transcriptional units of chromosome 1 of Leishmania major, extending on opposite strands from a point in the central region of the chromosome. A similar observation made on 93·4 kb of contiguous sequence for T. cruzi chromosome 3 suggests the presence of promoter and regulatory elements at the junctions of large polycistronic transcriptional units. All data obtained from the genome projects are made available through the public domain, which has prompted changing philosophies in how we approach analysis of the biology of these organisms, and strategies that we can employ now in the search for new therapies and vaccines.