Trypanosomatid Biology Research Articles

High-throughput prioritization of target proteins for development of new antileishmanial compounds

Leishmaniasis, a vector-borne disease, is caused by the infection of Leishmania spp., obligate intracellular protozoan parasites. Presently, human vaccines are unavailable, and the primary treatment relies heavily on systemic drugs, often presenting with suboptimal formulations and substantial toxicity, making new drugs a high priority for LMIC countries burdened by the disease, but a low priority in the agenda of most pharmaceutical companies due to unattractive profit margins. New ways to accelerate the discovery of new, or the repositioning of existing drugs, are needed. To address this challenge, our study aimed to identify potential protein targets shared among clinically-relevant Leishmania species. We employed a subtractive proteomics and comparative genomics approach, integrating high-throughput multi-omics data to classify these targets based on different druggability metrics. This effort resulted in the ranking of 6502 ortholog groups of protein targets across 14 pathogenic Leishmania species. Among the top 20 highly ranked groups, metabolic processes known to be attractive drug targets, including the ubiquitination pathway, aminoacyl-tRNA synthetases, and purine synthesis, were rediscovered. Additionally, we unveiled novel promising targets such as the nicotinate phosphoribosyltransferase enzyme and dihydrolipoamide succinyltransferases. These groups exhibited appealing druggability features, including less than 40% sequence identity to the human host proteome, predicted essentiality, structural classification as highly druggable or druggable, and expression levels above the 50th percentile in the amastigote form. The resources presented in this work also represent a comprehensive collection of integrated data regarding trypanosomatid biology.

Mitochondrial Ca2+ and Reactive Oxygen Species in Trypanosomatids.

Significance: Millions of people are infected with trypanosomatids and new therapeutic approaches are needed. Trypanosomatids possess one mitochondrion per cell and its study has led to discoveries of general biological interest. These mitochondria, as in their animal counterparts, generate reactive oxygen species (ROS) and have evolved enzymatic and nonenzymatic defenses against them. Mitochondrial calcium ion (Ca2+) overload leads to generation of ROS and its study could lead to relevant information on the biology of trypanosomatids and to novel drug targets. Recent Advances: Mitochondrial Ca2+ is normally involved in maintaining the bioenergetics of trypanosomes, but when Ca2+ overload occurs, it is associated with cell death. Trypanosomes lack key players in the mechanism of cell death described in mammalian cells, although mitochondrial Ca2+ overload results in collapse of their membrane potential, production of ROS, and cytochrome c release. They are also very resistant to mitochondrial permeability transition, and cell death after mitochondrial Ca2+ overload depends on generation of ROS. Critical Issues: In this review, we consider the mechanisms of mitochondrial oxidant generation and removal and the involvement of Ca2+ in trypanosome cell death. Future Directions: More studies are required to determine the reactions involved in generation of ROS by the mitochondria of trypanosomatids, their enzymatic and nonenzymatic defenses against ROS, and the occurrence and composition of a mitochondrial permeability transition pore. Antioxid. Redox Signal. 36, 969-983.

A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.

Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library's usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research.IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library's usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.

Identification of O-Glcnacylated Proteins in Trypanosoma cruzi.

Originally an anthropozoonosis in the Americas, Chagas disease has spread from its previous borders through migration. It is caused by the protozoan Trypanosoma cruzi. Differences in disease severity have been attributed to a natural pleomorphism in T. cruzi. Several post-translational modifications (PTMs) have been studied in T. cruzi, but to date no work has focused on O-GlcNAcylation, a highly conserved monosaccharide-PTM of serine and threonine residues mainly found in nucleus, cytoplasm, and mitochondrion proteins. O-GlcNAcylation is thought to regulate protein function analogously to protein phosphorylation; indeed, crosstalk between both PTMs allows the cell to regulate its functions in response to nutrient levels and stress. Herein, we demonstrate O-GlcNAcylation in T. cruzi epimastigotes by three methods: by using specific antibodies against the modification in lysates and whole parasites, by click chemistry labeling, and by proteomics. In total, 1,271 putative O-GlcNAcylated proteins and six modification sequences were identified by mass spectrometry (data available via ProteomeXchange, ID PXD010285). Most of these proteins have structural and metabolic functions that are essential for parasite survival and evolution. Furthermore, O-GlcNAcylation pattern variations were observed by antibody detection under glucose deprivation and heat stress conditions, supporting their possible role in the adaptive response. Given the numerous biological processes in which O-GlcNAcylated proteins participate, its identification in T. cruzi proteins opens a new research field in the biology of Trypanosomatids, improve our understanding of infection processes and may allow us to identify new therapeutic targets.

Thiol redox biology of trypanosomatids and potential targets for chemotherapy

Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different forms of leishmaniasis. This family of protozoan parasite possesses a trypanothione-based redox metabolism that provides the reducing equivalents for various vital processes such as the biosynthesis of DNA precursors and the detoxification of hydroperoxides. Almost all enzymes of the redox pathway proved to be essential and therefore fulfil one crucial prerequisite for a putative drug target. Trypanothione synthetase and trypanothione reductase are present in all trypanosomatids but absent from the mammalian host which, in addition to the essentiality, renders them highly specific. The chemotherapy research on both enzymes is further supported by the availability of high-throughput screening techniques and crystal structures. In this review we focus on the recent advances and limitations in the development of lead compounds targeting trypanothione synthetase and trypanothione reductase. We present an overview of the available inhibitors and discuss future perspectives including other components of the parasite-specific redox pathway.

Phytomonas: trypanosomatids adapted to plant environments.

Over 100 years after trypanosomatids were first discovered in plant tissues, Phytomonas parasites have now been isolated across the globe from members of 24 different plant families. Most identified species have not been associated with any plant pathology and to date only two species are definitively known to cause plant disease. These diseases (wilt of palm and coffee phloem necrosis) are problematic in areas of South America where they threaten the economies of developing countries. In contrast to their mammalian infective relatives, our knowledge of the biology of Phytomonas parasites and how they interact with their plant hosts is limited. This review draws together a century of research into plant trypanosomatids, from the first isolations and experimental infections to the recent publication of the first Phytomonas genomes. The availability of genomic data for these plant parasites opens a new avenue for comparative investigations into trypanosomatid biology and provides fresh insight into how this important group of parasites have adapted to survive in a spectrum of hosts from crocodiles to coconuts.

Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.

Actions of a proline analogue, L-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi.

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC50 = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Trypanosomatid Biology and Euglenozoan Evolution: New Insights and Shifting Paradigms Revealed through Genome Sequencing

Some of the biochemical peculiarities that occur in trypanosomatid protozoa are relatively well known: compartmentalisation of glycolytic enzymes within microbodies of the peroxisome class, an almost complete absence of gene regulation at the level of transcript initiation, and editing of mitochondrial transcripts. With the recent arrival of complete genome sequences for three distinct trypanosomatid parasites (Berriman et al. 2005; El-Sayed et al., 2005a and El-Sayed et al., 2005b; Ivens et al. 2005), new insights into the unusual biology of this fascinating family of protozoan parasites, considered to occupy one of the deeper branches of eukaryotic phylogeny, are apparent. Focusing mainly in the general areas of genome architecture and metabolism, this overview discusses some biological and evolutionary insights that have arisen as a consequence of sequence availability for the medically relevant trypanosomatids Trypanosoma brucei, T. cruzi, and Leishmania major. Much of the disease burden caused by these parasites occurs in the developing world, typically afflicting poor or isolated communities. In the future, a key translational application will be integration of the trypanosomatid genome sequences into ongoing discovery and development programmes for much needed new drugs

An organism-specific method to rank predicted coding regions in Trypanosoma brucei.

Genome annotation in differently evolved organisms presents challenges because the lack of sequence-based homology limits the ability to determine the function of putative coding regions. To provide an alternative to annotation by sequence homology, we developed a method that takes advantage of unusual trypanosomatid biology and skews in nucleotide composition between coding regions and upstream regions to rank putative open reading frames based on the likelihood of coding. The method is 93% accurate when tested on known genes. We have applied our method to the full complement of open reading frames on Chromosome I of Trypanosoma brucei, and we can predict with high confidence that 226 putative coding regions are likely to be functional. Methods such as the one described here for discriminating true coding regions are critical for genome annotation when other sources of evidence for function are limited.

Telomere biology of trypanosomatids: more questions than answers.

Trypanosomatids are severe pathogens in developing countries, where they affect both humans and domestic animals. Factors intrinsic to the host, the toxicity or subcurative effects of the available antiparasite medication and the low perspective of potential vaccines favor research on novel candidates for drug target. Telomeres are essential for the survival of most eukaryotes. In trypanosomatids, events such as antigenic variation and/or gene conversion and duplication occur at telomeric positions, possibly facilitating genome rearrangement. Understanding the role that telomere maintenance might play in the cell life span of trypanosomatids has important implications for therapeutics of parasitic diseases.

Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp.

Endotrypanum (order Kinetoplastida: family Trypanosomatidae) is a parasite of forest dwelling tree sloths (Edentata: genera Choleopus and Bradypus). Unique among the haemoflagellates, this protozoan has an intraerythrocytic phase in the mammalian host. Nevertheless, many striking similarities exist between Endotrypanum and the human pathogen Leishmania that make it a useful model for epidemiological and evolutionary aspects of the biology of trypanosomatids. Importantly, Endotrypanum species share both the insect vector and host reservoir with certain species of Leishmania (subgenus Viannia). Because mixed infections with Endotrypanum and Leishmania are common in sloths and, therefore, likely to occur in the sandfiy vector, there is a need for adequate biochemical markers to distinguish Endotrypanum from Leishmania infections. In this paper we show that Endotrypanum promastigotes possess sialidase and trans-sialidase activities, which are absent from Leishmania, and which are not closely related to the previously described trypanosomal sialidase/trans-sialidase enzyme. We also document the occurrence in Endotrypanum of homologues of the leishmanial surface metalloproteinase gp63 genes. The combined occurrence of sialidase/trans-sialidase activities and gp63 gene homologues in a unique organism has important ramifications for both field and laboratory studies on the biology of trypanosomatids, especially those related to host infection and evolution.

Herpetomonas megaseliae and Crithidia harmosa: Growth on Blood-Agar Plates

A technique for culturing two species of trypanosomatid flagellates on blood-agar plates is described. Herpetomonas megaseliae and Crithidia harmosa were grown as discrete clone colonies on plates prepared in sterile petri dishes with a medium derived from that of Tobie et al. (1950, J Parasitol 36: 48-54). Plates were inoculated with 0.01 ml flagellate suspension and the inoculum was spread evenly over the surface of the agar with a sterile glass spreader. Plates were incubated in a high humidity chamber at 25 C and colonies of both species, easily visible to the unaided eye, appeared within 1 week. There was a virtual 1:1 ratio between calculated number of CFU (colony forming units) and actual CFU observed on the plates. Colony morphology differed between the two species. Herpetomonas megaseliae produced a translucent flattened colony with a raised center and raised spiral arms, while C. harmosa produced an opaque hemispherical colony. Applications of the culture technique include potential diagnosis of trypanosomatid infections and potential studies in trypanosomatid genetics. Agar plates have been used for many years for the culture of microorganisms, and agarbased media have been used for nearly as long for culture of protozoa not normally grown on plates (Noller, 1917). Modifications of plating techniques have enabled workers to study the genetics of not only the cultured microorganisms, but also their parasites (Franklin et al., 1965; Lederberg and Lederberg, 1953; Puck, 1959). In the present paper we extend the technique of agar plate culture to include two protozoan species of the family Trypanosomatidae and suggest possible applications of the technique to problem areas in the study of trypanosomatid biology. MATERIALS AND METHODS Laboratory stock cultures of Herpetomonas megaseliae Daggett, Dollahon, and Janovy 1972 (ATCC # 30209) and Crithidia harmosa McGhee, Hanson, and Schmittner 1969, were used. The C. harmosa stock was obtained from Dr. S. H. Hutner, Pace University, and is from the same stock as ATCC # 30256. Organisms were loop transferred to 5-ml Mansour's medium with human blood (Dollahon and Janovy, 1971) and incubated at 25 C for 72 hr before use. Cultures used in these experiments were in the exponential phase of growth. Culture medium was prepared from the bloodagar base of that of Tobie et al. (1950) with the Locke's solution overlay salts added directly to the agar. Medium composition was thus: 10 g Bacto agar (Difco), 1.5 g Bacto beef (Difco), 2.5 g Neopeptone (Difco), 4 g NaCl, 0.1 g CaC2, 0.1 g KC1, 0.15 g NaH2PO4, 1.25 g glucose, and Received for publication 14 December 1976. 500 ml distilled water. This solution was boiled and adjusted to pH 7.2, autoclaved in a 1 liter screw cap flask, and allowed to cool until the flask was just warm to the touch. Forty ml of outdated whole human blood, lysed and diluted 1: 1 with distilled water, was added per 200 ml medium. Blood-agar plates were prepared in sterile glass petri dishes (15 by 100 mm) poured with 20 ml complete medium and allowed to solidify. Stock cultures of C. harmosa and H. megaseliae were counted by hemocytometer and diluted to contain the desired theoretical number of colony forming units (CFU) per 0.01 ml dilution medium. The dilution medium was Mansour's base (Dollahon and Janovy, 1971). Plates were then inoculated with 0.01 ml flagellate suspension and Giemsastained slides were also made of 0.01 ml inoculum suspension. The droplet on the agar plate was spread with a sterile Pasteur pipet with the tip bent at a right angle and sealed in a flame. Plates were sealed with masking tape, labeled, and placed in a high humidity chamber at 25 C. After 5 days growth, the numbers of colonies were counted and checked against the theoretical number of CFU inoculated. Inoculum slides were checked for differentiation state (of H. megaseliae) according to the methods of Janovy et al. (1975). The high humidity chamber consisted of plastic shoe boxes (10 by 15 by 30 cm) or aquaria (16 by 16 by 20 cm) with covers, containing several cotton balls soaked in water. All inoculation procedures were carried out in an isolation hood sterilized by 24-hr continuous UV lighting. Smears of organisms were air dried, fixed in absolute methanol, and Giemsa-stained. Stain was diluted 1:50 with phosphate buffer at pH 7.2.