Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

How are Trypanosoma brucei receptors protected from host antibody-mediated attack?

Trypanosoma brucei is the causal agent of African Trypanosomiasis in humans and other animals. It maintains a long-term infection through an antigenic variation based population survival strategy. To proliferate in a mammal, T. brucei acquires iron and haem through the receptor mediated uptake of host transferrin and haptoglobin-hemoglobin respectively. The receptors are exposed to host antibodies but this does not lead to clearance of the infection. Here we discuss how the trypanosome avoids this fate in the context of recent findings on the structure and cell biology of the receptors.

Antimalarial evaluation of alkyl-linked bis-thiadiazine derivatives in murine model infected with two Plasmodium strains.

Background and Purpose: Plasmodium falciparum and P. vivax are responsible for most malaria cases in humans in the African Region and the Americas; these parasites have developed resistance to classic antimalarial drugs. On the other hand, previous investigations of the alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) derivatives compounds show satisfactory results against protozoan parasites such as Trypanosoma cruzi, Trypanosoma vaginalis, Trypanosoma brucei rhodesiense and Leishmania donovani. Therefore, it is possible to see some effect of bis-THTT derivatives on other protozoan parasites, such as Plasmodium. Experimental Approach: This study aimed to perform an in vivo biological evaluation of bis-THTT (JH1 to JH6) derivatives compounds as possible anti-malaria drugs in BALB/c mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL strains. In this work, we evaluated the compounds as potential antimalarial drugs in BALB/c mice infected with Plasmodium strains. Key Results: For each compound, we assess the percentages of parasitemia by smears from tail blood and the humoral response by indirect ELISA test using each compound as an antigen. We also evaluated the B lymphocyte response and the cytotoxicity of the bis-THTT derivatives compounds with MTT cell proliferation assays. Conclusions: Our results show that the bis-THTT derivatives JH2 and JH4 presented effective parasitemia control in mice infected with P. berghei; JH5 and JH6 compounds have similar infection control results as chloroquine in mice infected P. yoelii strain. The evaluation of bis-THTT derivatives compounds in a model of BALB/c mice infected with P. berghei and P. yoelii allowed us to conclude that some of them have an antimalarial effect; however, none of the tested compounds exceeded the efficiency of chloroquine.

Comparison of Three Widely Employed Extraction Methods for Metabolomic Analysis of Trypanosoma brucei.

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites' physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei.

Discovery of 1,3,4-Oxadiazole Derivatives as Broad-Spectrum Antiparasitic Agents.

Vector-borne parasitic diseases (VBPDs) pose a significant threat to public health on a global scale. Collectively, Human African Trypanosomiasis (HAT), Leishmaniasis, and Malaria threaten millions of people, particularly in developing countries. Climate change might alter the transmission and spread of VBPDs, leading to a global burden of these diseases. Thus, novel agents are urgently needed to expand therapeutic options and limit the spread of drug-resistant parasites. Herein, we report the development of broad-spectrum antiparasitic agents by screening a known library of antileishmanial and antimalarial compounds toward Trypanosoma brucei (T. brucei) and identifying a 1,3,4-oxadiazole derivative (19) as anti-T. brucei hit with predicted blood-brain barrier permeability. Subsequently, extensive structure-activity-relationship studies around the lipophilic tail of 19 led to a potent antitrypanosomal and antimalarial compound (27), with moderate potency also toward Leishmania infantum (L. infantum) and Leishmania tropica. In addition, we discovered a pan-active antiparasitic molecule (24), showing low-micromolar IC50s toward T. brucei and Leishmania spp. promastigotes and amastigotes, and nanomolar IC50 against Plasmodium falciparum, together with high selectivity for the parasites over mammalian cells (THP-1). Early ADME-toxicity assays were used to assess the safety profile of the compounds. Overall, we characterized 24 and 27, bearing the 1,3,4-oxadiazole privileged scaffold, as broad-spectrum low-toxicity agents for the treatment of VBPDs. An alkyne-substituted chemical probe (30) was synthesized and will be utilized in proteomics experiments aimed at deconvoluting the mechanism of action in the T. brucei parasite.

Transcriptome profiles of Trypanosoma brucei rhodesiense in Malawi reveal focus specific gene expression profiles associated with pathology.

BACKGROUND: Sleeping sickness caused by Trypanosoma brucei rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi. METHODOLOGY: Blood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNP calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure. RESULTS: 24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a less symptomatic disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. CONCLUSION: Our results suggest that the differences in disease presentation in the two foci is mainly driven by genetic differences in the parasites in the two major endemic foci of Rumphi and Nkhotakota rather than differences in the environment or host response.

Assessment of optimal blood exposure time at 37 °C during in vitro tsetse flies feeding for quality production in mass-rearing colonies.

Control of African animal trypanosomosis is implemented through an integrated control strategy, with the sterile insect technique (SIT) as one of its components. The SIT requires mass rearing of tsetse fly colonies using an in vitro feeding system. The exposure of blood at 37 °C on heating plates over time can have an impact on the quality of fly productivity. In this study, we investigated the survival and fecundity of adult tsetse flies fed at 37 °C on 8 blood exposure times ranging from 30 min to 4 h with increments of 30 min (treatment 1, flies were fed 30 min after exposure to blood at 37 °C; treatment 2, 1 h and so on until treatment 8 [4 h after]) in order to determine the optimal exposure time. In addition, bacterial growth in blood from each treatment was assessed by agar culture at 37 °C for 72 h. The results showed that the adult female survival rates were similar regardless of the treatment. For males, only those of treatment 1 (30 min) showed a marginal lower survival than those of treatments 7 and 8 fed after 3 h 30 min and 4 h of blood exposure, respectively. Over the 4-h interval of blood exposure at 37 °C, the results showed that the number of pupae produced per initial female and pupal weight tended to increase with exposure time, but the differences were not significant. We discuss the implications of these results on tsetse mass rearing for the SIT program.

Anogeissus leiocarpus (DC.) Guill. & Perr. (Combretaceae): A review of the traditional uses, phytochemistry and pharmacology of African birch.

Anogeissus leiocarpus (DC.) Guill. & Perr. belongs to the family Combretaceae and is used both by African traditional medical practitioners and livestock rearers to treat diseases such as African trypanosomiasis, animal diarrhoea, asthma, cancer, cough, diabetes, dysentery, erectile dysfunction, fever, giardiasis, helminthiases, meningitis, menstrual disorders, monkeypox, oral infections, poliomyelitis, sickle cell anaemia, snake bites, toothache, urinary schistosomiasis, and yellow fever. Some of these activities have been associated with the presence of polyphenols in the plant which include ellagic acid derivatives, flavonoids, stilbenes, tannins, and triterpenes. Several bioactive molecules have been identified from A. leiocarpus. These include the main active constituents, ellagitannins, ellagic acid derivates, flavonoids and triterpenes. Pharmacological studies have confirmed its antibacterial, antifungal, antihyperglycemic, antihypertensive, antimalarial, antioxidative, antiparasitic, antitumour and anti-ulcer effects. The stem bark has been investigated mainly for biological activities and phytochemistry, and it is the most mentioned plant part highlighted by the traditional users in ethnomedicinal surveys. In vitro and in vivo models, which revealed a wide range of pharmacological actions against parasites causing helminthiasis, leishmaniasis, malaria and trypanosomiasis, have been used to study compounds from A. leiocarpus. Because of its uses in African traditional medicine and veterinary practices, A. leiocarpus has received considerable attention from researchers. The current review provides a comprehensive overview and critical appraisal of scientific reports on A. leiocarpus, covering its traditional uses, pharmacological activities and phytochemistry.

The Transmission of Animal African Trypanosomiasis in Two Districts in the Forest Zone of Ghana.

Animal African trypanosomiasis, also known as nagana, is caused by Trypanosoma species, which cause significant clinical diseases and lead to losses in animal production. We carried out a cross-sectional survey to investigate the composition of vectors and parasite diversity in two districts in the eastern region of Ghana where pigs and cattle were exposed to tsetse bites. We performed cytochrome c oxidase subunit 1 polymerase chain reaction (PCR) to identify tsetse species and internal transcribed spacer 1 PCR to identify Trypanosoma species. Also, we investigated the source of tsetse blood meal based on mitochondrial cytochrome b gene sequence analysis. A total of 229 tsetse, 65 pigs, and 20 cattle were investigated for trypanosomes. An overall vector density of 4.3 tsetse/trap/day was observed. A trypanosome prevalence of 58.9% (95% CI = 52.5-65.1%), 46.2% (95% CI = 34.6-58.1%), and 0.0% (95% CI = 0.0-16.1%) in tsetse, pigs, and cattle, respectively, was detected. Trypanosoma congolense was predominant, with a prevalence of 33.3% (95% CI = 73.3-86.5%) in tsetse. There was evidence of multiple infections in tsetse and pigs. Approximately 39% of the tsetse were positive for multiple infections of T. congolense and Trypanosoma simiae. Parasite prevalence in pigs across the communities was high, with significant differences associated between locations (χ2 = 28.06, 95% CI = 0.05-0.81, P = 0.0009). Tsetse blood meal analysis revealed feeding on domestic Sus scrofa domesticus (pigs) and Phacochoerus africanus (warthogs). Infective tsetse may transmit trypanosomes to livestock and humans in the communities studied.

From pole to pole, life-long research of sleep in extreme environments.

In November 1965, Michel Jouvet accepted me into his laboratory in Lyon as a medical student at a time when sleep research was an adventure. After 4 years of investigations in cats, I obtained my medical doctorate. Being a military physician, I was posted to Antarctica for wintering over and was initiated by Jean Rivolier into the psychology of small isolated human groups. I recorded 180 polysomnographic (PSG) nights in eight of my companions. This was my first contribution to research on human sleep under extreme environments and conditions. I then entered René Hénane's military thermophysiology laboratory, where I analyzed thermal exchanges during human sleep in the heat. Back to the cold, I spent 2 years in Canada and analyzed sleep during the Arctic winter under the direction of Manny W. Radomski, who headed the Defense and Civil Institute of Environmental Medicine and judged my PhD dissertation along with my first two mentors. Throughout my career, I worked in collaboration with Manny Radomski under the auspices of the Franco-Canadian Accord for Defence Research. We studied sleep and exercise, sleep deprivation, and recovery with and without chemical help. He also gave me support during several investigations in Africa. There, I studied normal sleep under various tropical climates (warm and dry in Niger, warm and humid in Côte d'Ivoire and Congo, temperate mid-mountain in Angola). I determined that human African trypanosomiasis, the ravaging sleeping sickness or tsetse disease, is not a hypersomnia, but a disorder of circadian rhythms, notably in the sleep-wake cycle.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

m6A landscape is more pervasive when Trypanosoma brucei exits the cell cycle.

N6-methyladenosine (m6A) is a mRNA modification with important roles in gene expression. In African trypanosomes, this post-transcriptional modification is detected in hundreds of transcripts and it affects the stability of the variant surface glycoprotein (VSG) transcript in the proliferating blood stream form. However, how the m6A landscape varies across the life cycle remains poorly defined. Using full-length, non-fragmented RNA, we immunoprecipitated and sequenced m6A-modified transcripts across three life cycle stages of Trypanosoma brucei - slender (proliferative), stumpy (quiescent), and procyclic forms (proliferative). We found that 1037 transcripts are methylated in at least one of these three life cycle stages. While 21% of methylated transcripts are common in the three stages of the life cycle, globally each stage has a distinct methylome. Interestingly, 47% of methylated transcripts are detected in the quiescent stumpy form only, suggesting a critical role for m6A when parasites exit the cell cycle and prepare for transmission by the Tsetse fly. In this stage, we found that a significant proportion of methylated transcripts encodes for proteins involved in RNA metabolism, which is consistent with their reduced transcription and translation. Moreover, we found that not all major surface proteins are regulated by m6A, as procyclins are not methylated, and that, within the VSG repertoire, not all VSG transcripts are demethylated upon parasite differentiation to procyclic form. This study reveals that the m6A regulatory landscape is specific to each life cycle stage, becoming more pervasive as T. brucei exits the cell cycle.

A lineage-specific protein network at the trypanosome nuclear envelope.

The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.

Privileged small molecules against neglected tropical diseases: A perspective from structure activity relationships.

Neglected tropical diseases (NTDs) comprise diverse infections with more incidence in tropical/sub-tropical areas. In spite of preventive and therapeutic achievements, NTDs are yet serious threats to the public health. Epidemiological reports of world health organization (WHO) indicate that more than 1.5 billion people are afflicted with at least one NTD type. Among NTDs, leishmaniasis, chagas disease (CD) and human African trypanosomiasis (HAT) result in substantial morbidity and death, particularly within impoverished countries. The statistical facts call for robust efforts to manage the NTDs. Currently, most of the anti-NTD drugs are engaged with drug resistance, lack of efficient vaccines, limited spectrum of pharmacological effect and adverse reactions. To circumvent the issue, numerous scientific efforts have been directed to the synthesis and pharmacological development of chemical compounds as anti-infectious agents. A survey of the anti-NTD agents reveals that the majority of them possess privileged nitrogen, sulfur and oxygen-based heterocyclic structures. In this review, recent achievements in anti-infective small molecules against parasitic NTDs are described, particularly from the SAR (Structure activity relationship) perspective. We also explore current advocating strategies to extend the scope of anti-NTD agents.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.

To Explore Potential Inhibitors Against Various Enzymatic Targets of Human African Trypanosomiasis.

Synthetic drugs currently prescribed for the treatment of Human African Trypanosomiasis (HAT) are non-specific, toxic, demand extended therapeutic regimes and are of varying efficacy. Along with the challenging demographic and socio-economic hurdles, the everincreasing risk of drug resistance is another major problem to be addressed. Cysteine protease, Heat shock proteins (HSP-90), Trypanothione reductase (TR), Farnesyl diphosphate synthase, Glucose-6-phosphate dehydrogenase, UP-4-galactose epimerase, and Cytidine triphosphate synthetase are potential enzymatic targets for the development of novel inhibitors against HAT which are the main focus of this review. The potential enzymatic targets of Trypanosoma brucei, especially small molecules like cysteine proteases and heat shock proteins are identified as major candidates for the sustenance of the parasite, their proliferation, infection, and spread of the disease. The development of new compounds to combat the disease by thorough ligand modification has been explored in the current review. Extracting these compounds and studying their efficacy, toxicity, and target mechanism extensively, this review has proposed a list of different compounds, including some synthetic and natural compounds along with multi-target inhibitors such as acoziborole, fexinidazole, etc. Potential inhibitors against these enzymatic targets of the T. brucei are important candidates for designing novel therapeutics against HAT. Multi-target inhibitors have also been identified as crucial molecules because of their potential advantage against the development of drug resistance.

Modelled impact of Tiny Targets on the distribution and abundance of riverine tsetse.

BACKGROUND: The insecticide-treated baits known as Tiny Targets are one of the cheapest means of controlling riverine species of tsetse flies, the vectors of the trypanosomes that cause sleeping sickness in humans. Models of the efficacy of these targets deployed near rivers are potentially useful in planning control campaigns and highlighting the principles involved. METHODS AND PRINCIPAL FINDINGS: To evaluate the potential of models, we produced a simple non-seasonal model of the births, deaths, mobility and aging of tsetse, and we programmed it to simulate the impact of seven years of target use against the tsetse, Glossina fuscipes fuscipes, in the riverine habitats of NW Uganda. Particular attention was given to demonstrating that the model could explain three matters of interest: (i) good control can be achieved despite the degradation of targets, (ii) local elimination of tsetse is impossible if invasion sources are not tackled, and (iii) with invasion and target degradation it is difficult to detect any effect of control on the age structure of the tsetse population. CONCLUSIONS: Despite its simplifications, the model can assist planning and teaching, but allowance should be made for any complications due to seasonality and management challenges associated with greater scale.

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes.

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.