Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics.

African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single-cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated (CCR) transcriptomes of both procyclic and slender BSF Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single-cell transcriptomic analysis of cryopreserved T. brucei. Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the CCR transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms.

Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host-parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

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.

The IP3 receptor and Ca2+ signaling in trypanosomes.

Trypanosoma cruzi, and the T. brucei group of parasites cause neglected diseases that affect millions of people around the world. These unicellular microorganisms have complex life cycles involving an insect vector and a mammalian host. Both groups of pathogens possess an inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) signaling pathway, and an IP3 receptor, but with lineage-specific adaptations that make them different from their mammalian counterparts. The phospholipase C (PLC), which hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to IP3 is N-terminally myristoylated and palmitoylated. Acidocalcisomes, which are lysosome-related organelles rich in polyphosphate, are the main intracellular Ca2+ stores. The inositol 1,4,5-trisphosphate receptor (IP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. The trypanosome IP3R is stimulated by luminal phosphate and pyrophosphate, which are hydrolysis products of polyphosphate (polyP), and inhibited by tripolyphosphate (polyP3), which is the most abundant polyP in acidocalcisomes. Ca2+ signaling is important for host cell invasion and differentiation and to maintain cellular bioenergetics.

Evolving Differentiation in African Trypanosomes.

Differentiation is a central aspect of the parasite life cycle and encompasses adaptation to both host and environment. If we accept that evolution cannot anticipate an organism's needs as it enters a new environment, how do parasite differentiation pathways arise? The transition between vertebrate and insect stage African trypanosomes is probably one of the better studied and involves a cell-cycle arrested or 'stumpy' form that activates metabolic pathways advantageous to the parasite in the insect host. However, a range of stimuli and stress conditions can trigger similar changes, leading to formation of stumpy-like cellular states. We propose that the origin and optimisation of this differentiation program represents repurposing of a generic stress response to gain considerable gain-of-fitness associated with parasite transmission.

Nanotechnology enabled the enhancement of antitrypanosomal activity of piperine against Trypanosoma evansi.

Nanoencapsulation is the promising approach to enhance the therapeutic potential of a drug. In the present investigation, piperine-loaded nanocapsules (NCs) was prepared and evaluated for antitrypanosomal activity against the parasite Trypanosoma evansi, a causative agent of trypanosomiasis. Piperine, a bioactive compound was selected as an alternative for drugs that have been used for the treatment of the disease from decades to overcome the toxic effects or drug resistance effect. Moreover, piperine has reported to possess therapeutic potential against other Trypanosoma spp. and has also been reported to cause reactive oxygen species (ROS) mediated effect in cancer cells that was the other reason for the selection. To date, piperine and its nanoformulations have not been evaluated for their growth inhibitory effect against T. evansi. Piperine-loaded NCs exhibited more significant antitrypanosomal effect at approximately three-times less IC50 value 5.04 µM as compared to piperine (IC50-14.45 µM). Moreover, increased production of reactive oxygen species observed in the case of piperine-loaded NCs as that of pure piperine in the axenic culture of T. evansi. Furthermore, different concentrations of piperine-loaded NCs showed less cytotoxicity on horse peripheral blood mononuclear cells as liken to pure piperine. In conclusion, our results demonstrated that piperine-loaded NCs induced more generation of ROS that contributed inhibitory effect on the growth of Trypanosoma evansi as compared to pure drug.

Drug-induced reactive oxygen species-mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 µM and 0.33 µM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Morphological and molecular characterization of an African freshwater fish trypanosome, including its development in a leech vector.

Trypanosomes are ubiquitous blood parasites of fishes and at least 16 species were originally described infecting African freshwater fishes. This number was later reduced to six and in the late 1990s it was proposed that most records of freshwater fish trypanosomes across Africa are Trypanosoma mukasai Hoare, 1932. Recently, results from a molecular analysis of fish trypanosomes from the Okavango Delta, Botswana, reported the presence of at least two genotypic groups and concluded that the identification of T. mukasai remains problematic. The aims of the present study were thus to elucidate the life cycle of a freshwater fish trypanosome from southern Africa and to do a morphological and molecular characterization of this parasite from both the fish host and leech vector. To locate trypanosome stages, leeches were removed from fishes captured in the Phongolo River, South Africa, and fish blood films and leech squashes were Giemsa-stained and screened. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted and fragments of the 18S rDNA gene were amplified and sequenced. Trypanosomes were detected in the fish families Cichlidae, Clariidae, Mochokidae and Schilbeidae. Sequence data showed that the trypanosome from one of the leeches, identified as Batracobdelloides tricarinata (Blanchard, 1897), was highly similar to those obtained from the plain squeaker, Synodontis zambezensis, with 0.7% difference recorded between them. From morphological and molecular data presented here, it is clear that the trypanosomes from Phongolo are closely related to those of the Okavango and should be considered as a single diverse species with genetic differentiation between 0.4-2.9%, under the 3-5% differences expected to be seen between true distinct species within the rRNA. Developmental stages of the trypanosome found in the leech B. tricarinata supports its status as the vector and the molecular evidence shows the relationship between the trypanosome in the fish and leech, but also illustrates the exceptional genetic and morphological diversity of a single species of trypanosome between host species. The work presented here provides us with clear information to take further steps in resolving the taxonomy and systematics of African freshwater fish trypanosomes.

Development of Trypanosoma everetti in Culicoides biting midges.

Trypanosoma species (Trypanosomatida, Kinetoplastea) are almost exclusively heteroxenous flagellated parasites, which have been extensively studied as the causative agents of severe trypanosomiasis in humans and domestic animals. However, the biology of avian trypanosomes remains insufficiently known, particularly in wildlife, despite information that some species might be pathogenic and affect the fitness of intensively infected individuals. Avian trypanosomes are cosmopolitans. Due to regular bird seasonal migrations, this host-parasite system might provide new insight for better understanding mechanisms of transcontinental dispersal of pathogens, their ecological plasticity, specificity and speciation. Trypanosoma everetti parasitizes numerous bird species globally, but data on its biology are scarce and its vectors remain unknown. This study aimed to test experimentally whether widespread Culicoides (Diptera: Ceratopogonidae) biting midges are susceptible to infection with this parasite. Two common house martins Delichon urbicum and two sedge warblers Acrocephalus schoenobaenus naturally infected with T. everetti were caught in the wild after arrival from African wintering grounds. Laboratory reared Culicoides nubeculosus and wild-caught Culicoides impunctatus biting midges were exposed by allowing them to take infected blood meals. The experimentally infected and control insects were maintained in the laboratory and dissected at intervals to follow the development of the parasite. Infections were determined using microscopic examination and PCR-based testing. Four closely related haplotypes of T. everetti were found, and each was present in different individual parasite-donor birds. These parasites readily developed and produced metacyclic trypomastigotes in C. nubeculosus and C. impunctatus biting midges. Molecular characterisation of T. everetti was developed. According to Bayesian phylogenetic analysis using a DNA fragment encoding 18S rRNA, the five species of small avian trypanosomes were closely related. Wild caught Culicoides biting midges were also collected and screened for the presence of natural infections. In all, 6.8% of wild-caught biting midges belonging to five Culicoides species were PCR-positive for kinetoplastids, including Trypanosoma species. Culicoides biting midges are readily susceptible and likely naturally transmit avian trypanosomes and thus, should be targeted in epidemiology research of avian trypanosomiasis.

A Leap Into the Unknown - Early Events in African Trypanosome Transmission.

African trypanosomes are mainly transmitted by tsetse flies. In recent years there has been good progress in understanding how the parasites prepare for transmission, detect their changed environment through the perception of different environmental cues, and respond by changing their developmental gene expression. In this review, we discuss the different signals and signaling mechanisms used by the parasites to carry out the early events necessary for their establishment in the fly. We also compare Trypanosoma brucei and Trypanosoma congolense, parasites that share a common pathway in the early stages of fly colonization but apparently use different mechanisms to achieve this.

Phylogenetics, patterns of genetic variation and population dynamics of Trypanosoma terrestris support both coevolution and ecological host-fitting as processes driving trypanosome evolution.

BACKGROUND: A considerable amount of evidence has favored ecological host-fitting, rather than coevolution, as the main mechanism responsible for trypanosome divergence. Nevertheless, beyond the study of human pathogenic trypanosomes, the genetic basis of host specificity among trypanosomes isolated from forest-inhabiting hosts remains largely unknown. METHODS: To test possible scenarios on ecological host-fitting and coevolution, we combined a host capture recapture strategy with parasite genetic data and studied the genetic variation, population dynamics and phylogenetic relationships of Trypanosoma terrestris, a recently described trypanosome species isolated from lowland tapirs in the Brazilian Pantanal and Atlantic Forest biomes. RESULTS: We made inferences of T. terrestris population structure at three possible sources of genetic variation: geography, tapir hosts and 'putative' vectors. We found evidence of a bottleneck affecting the contemporary patterns of parasite genetic structure, resulting in little genetic diversity and no evidence of genetic structure among hosts or biomes. Despite this, a strongly divergent haplotype was recorded at a microgeographical scale in the landscape of Nhecolândia in the Pantanal. However, although tapirs are promoting the dispersion of the parasites through the landscape, neither geographical barriers nor tapir hosts were involved in the isolation of this haplotype. Taken together, these findings suggest that either host-switching promoted by putative vectors or declining tapir population densities are influencing the current parasite population dynamics and genetic structure. Similarly, phylogenetic analyses revealed that T. terrestris is strongly linked to the evolutionary history of its perissodactyl hosts, suggesting a coevolving scenario between Perissodactyla and their trypanosomes. Additionally, T. terrestris and T. grayi are closely related, further indicating that host-switching is a common feature promoting trypanosome evolution. CONCLUSIONS: This study provides two lines of evidence, both micro- and macroevolutionary, suggesting that both host-switching by ecological fitting and coevolution are two important and non-mutually-exclusive processes driving the evolution of trypanosomes. In line with other parasite systems, our results support that even in the face of host specialization and coevolution, host-switching may be common and is an important determinant of parasite diversification.

Trypanosoma evansi impacts on embryonic neural progenitor cell functions.

Trypanosoma evansi appears to have a significant tropism for brain tissue in its chronic and acute phases. The most common symptoms of this brain infection are motor incoordination, meningoencephalitis, demyelination, and anemia. There have only been few studies of the effects of T. evansi infection on neuronal differentiation and brain plasticity. Here, we investigated the impact of the congenital T. evansi infection on brain development in mice. We collected telencephalon-derived neural progenitor cells (NPCs) from T. evansi uninfected and infected mice, and cultivated them into neurospheres. We found that T. evansi significantly decreased the number of cells during development of neurospheres. Analysis of neurosphere differentiation revealed that T. evansi infection significantly increased neural migration. We also observed that T. evansi promoted expression of glial fibrillary acidic protein (GFAP) in infected cells. These data suggest that congenital T. evansi infection may affect embryonic brain development.

The early-acting glycosome biogenic protein Pex3 is essential for trypanosome viability.

Trypanosomatid parasites are infectious agents for diseases such as African sleeping sickness, Chagas disease, and leishmaniasis that threaten millions of people, mostly in the emerging world. Trypanosomes compartmentalize glycolytic enzymes to an organelle called the glycosome, a specialized peroxisome. Functionally intact glycosomes are essential for trypanosomatid viability, making glycosomal proteins as potential drug targets against trypanosomatid diseases. Peroxins (Pex), of which Pex3 is the master regulator, control glycosome biogenesis. Although Pex3 has been found throughout the eukaryota, its identity has remained stubbornly elusive in trypanosomes. We used bioinformatics predictive of protein secondary structure to identify trypanosomal Pex3. Microscopic and biochemical analyses showed trypanosomal Pex3 to be glycosomal. Interaction of Pex3 with the peroxisomal membrane protein receptor Pex19 observed for other eukaryotes is replicated by trypanosomal Pex3 and Pex19. Depletion of Pex3 leads to mislocalization of glycosomal proteins to the cytosol, reduced glycosome numbers, and trypanosomatid death. Our findings are consistent with Pex3 being an essential gene in trypanosomes.

Regulation of gene expression in trypanosomatids: living with polycistronic transcription.

In trypanosomes, RNA polymerase II transcription is polycistronic and individual mRNAs are excised by trans-splicing and polyadenylation. The lack of individual gene transcription control is compensated by control of mRNA processing, translation and degradation. Although the basic mechanisms of mRNA decay and translation are evolutionarily conserved, there are also unique aspects, such as the existence of six cap-binding translation initiation factor homologues, a novel decapping enzyme and an mRNA stabilizing complex that is recruited by RNA-binding proteins. High-throughput analyses have identified nearly a hundred regulatory mRNA-binding proteins, making trypanosomes valuable as a model system to investigate post-transcriptional regulation.

Morphological and molecular characterization of avian trypanosomes in raptors from Thailand.

From September 2012 to May 2018, blood samples from 364 raptors (mostly adults) were collected and screened for trypanosomes and haemosporidians by microscopic examination and nested polymerase chain reactions (PCR). Trypanosoma spp. were identified in 15 birds from eight different species. Light microscopy revealed 14 cases of infection with Trypanosoma cf. corvi, including one each in black-shouldered kite (Elanus caeruleus, n = 49), Brahminy kite (Haliastur indus, n = 50), and spotted owlet (SO, Athene brama, n = 27); two mountain hawk-eagles (Spizaetus nipalensis, n = 3); and three each in Asian barred owlets (ABO, Glaucidium cuculoides, n = 27), barn owls (BO, Tyto alba, n = 65) and collared scops owls (CSO, Otus lettia, n = 41). In addition, one case of infection with T. avium was identified in an oriental scops owl (OSO, Otus sunia, n = 2). All infected raptors showed very low parasitemia levels. The PCR detected more three positives in one CSO, one Japanese sparrowhawk (Accipiter gularis), and one OSO. The sensitivity and specificity of the PCR method were 93.3% and 99.1%, respectively. The overall infection rate was very low (4.9%). The highest infection rate was recorded in cold-dry season (9.9%). Coinfection of Plasmodium with trypanosomes was found in all three ABOs. Coinfection with Haemoproteus spp. was found in one BO, three CSOs, and one SO. Coinfection with Haemoproteus spp. and Leucocytozoon danilewskyi was found in the OSO. Microfilarias were detected in one ABO and one CSO. The ultrastructure of trypomastigotes of T. cf. corvi in an ABO revealed fine structures. All small subunit ribosomal RNA (SSU rRNA) sequences belong to two clades: T. avium and T. corvi-culicavium complex/group. SSU rRNA gene amplification was not successful in one BO. The raptors with trypanosome infections showed normal hematological values and healthy appearance. Furthermore, this is the first report of T. avium in a nocturnal raptor from Thailand.

The marsupial trypanosome Trypanosoma copemani is not an obligate intracellular parasite, although it adversely affects cell health.

BACKGROUND: Trypanosoma cruzi invades and replicates inside mammalian cells, which can lead to chronic Chagas disease in humans. Trypanosoma copemani infects Australian marsupials and recent investigations indicate it may be able to invade mammalian cells in vitro, similar to T. cruzi. Here, T. cruzi 10R26 strain (TcIIa) and two strains of T. copemani [genotype 1 (G1) and genotype 2 (G2)] were incubated with marsupial cells in vitro. Live-cell time-lapse and fluorescent microscopy, combined with high-resolution microscopy (transmission and scanning electron microscopy) were used to investigate surface interactions between parasites and mammalian cells. RESULTS: The number of parasites invading cells was significantly higher in T. cruzi compared to either genotype of T. copemani, between which there was no significant difference. While capable of cellular invasion, T. copemani did not multiply in host cells in vitro as there was no increase in intracellular amastigotes over time and no release of new trypomastigotes from host cells, as observed in T. cruzi. Exposure of host cells to G2 trypomastigotes resulted in increased host cell membrane permeability within 24 h of infection, and host cell death/blebbing was also observed. G2 parasites also became embedded in the host cell membrane. CONCLUSIONS: Trypanosoma copemani is unlikely to have an obligate intracellular life-cycle like T. cruzi. However, T. copemani adversely affects cell health in vitro and should be investigated in vivo in infected host tissues to better understand this host-parasite relationship. Future research should focus on increasing understanding of the T. copemani life history and the genetic, physiological and ecological differences between different genotypes.

Biological study of Trypanosoma caninum under co-culture with different feeder layer cells.

Trypanosoma caninum is a parasite isolated from domestic dogs, of which several biological aspects remain unknown, including evolutive forms found in vertebrate hosts. The objective of this study was to evaluate co-cultures of T. caninum with different cell lines as feeder layers to monitor the differentiation process and investigate infective potential. The study was performed using DH-82, MDCK, and Lulo cell lines. T. caninum from axenic culture was added to the cultured adherent cells. At intervals over 30 days, aliquots of the supernatant were collected for quantification and assessment of differentiation. Infectivity assays were performed on the aforementioned cell lines seeded on glass coverslips and evaluated after 6, 24, and 72 h. In the supernatant of the feeder layer, T. caninum presented similar growth profiles, with epimastigote and trypomastigote forms in binary and multiple divisions. During co-culture with DH-82 and MDCK cells, a higher level of differentiation to trypomastigotes was observed. This study shows that the differentiation process of this parasite can vary according to culture conditions and that DH-82 and MDCK lineages could be applied to the study of trypomastigote forms. All forms of T. caninum described until now (aflagellar epimastigotes, typical epimastigotes, or trypomastigotes) were unable to infect the cell line Finally, this study provides additional data about morphobiological aspects. Although the biological cycle of T. caninum has not been established, the present data suggest the importance of feeder layers in promoting the growth and differentiation of this new parasite.

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

Antiparasitic activity against trypanosomatid diseases and novel metal complexes derived from the first time characterized 5-phenyl-1,2,4-triazolo[1,5-a]pyrimidi-7(4H)-one.

A serie of isostructural complexes with general formula [M(ftpO)2(H2O)4] have been obtained from reaction between the first time characterized triazolopyrimidine derivative 5-phenyl-1,2,4-triazolo[1,5-a]pyrimidi-7(4H)-one (HftpO) (1) and first row transition nitrates (M=Cu (2), Co (3), Ni (4) and Zn (5)). A copper complex with formula [Cu(HftpO)2(NO3)2(H2O)2]·H2O (6) was also isolated. HftpO and their metal complexes have been characterized by spectroscopic and thermal analysis and their crystal structures have been solved by X-ray diffraction methods. The isostructural compounds are mononuclear complexes where the triazolopyrimidine ligand acts as monodentate ligand through N3 nitrogen position. The crystal structure of these novel bis-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-tetraaquo metal complexes offers an excellent opportunity at these complexes to acts as potential building blocks. Also, the antiparasitic activity of HftpO ligand against different leishmania and trypanosome strains has been studied.